Substrates, Systems, and Methods for Array Synthesis and Biomolecular Analysis

ABSTRACT

Disclosed herein are formulations, substrates, and arrays. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays, including porous arrays. Also disclosed herein are formulations and methods for one-step coupling, e.g., for synthesis of peptides in an N-&gt;C orientation. In some embodiments, disclosed herein are formulations and methods for high efficiency coupling of biomolecules to a substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/287,968, filed Feb. 27, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/442,389, filed May 12, 2015, which issued asU.S. Pat. No. 10,286,376, which is the National Stage of InternationalPatent Application No. PCT/US/2013/070207, filed Nov. 14, 2013, whichclaims the benefit of U.S. Provisional Patent Application No. 61/726,515filed Nov. 14, 2012, U.S. Provisional Patent Application No. 61/732,221,filed Nov. 30, 2012, U.S. Provisional Patent Application No. 61/805,884,filed Mar. 27, 2013, U.S. Provisional Patent Application No. 61/765,584,filed Feb. 15, 2013, U.S. Provisional Patent Application No. 61/866,512,filed Aug. 15, 2013; PCT/US2013/070207 is also a continuation-in-part ofInternational Patent Application No. PCT/US2013/062773, filed Sep. 30,2013, which is a continuation-in-part of PCT/US2013/025190, filed Feb.7, 2013, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/761,347, filed Feb. 6, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 14, 2020, is namedVIB-007C2_sequence_listing.txt, and is 3,605 bytes in size.

BACKGROUND

A typical microarray system is generally comprised of biomolecularprobes, such as DNA, proteins, or peptides, formatted on a solid planarsurface like glass, plastic, or silicon chip, plus the instrumentsneeded to handle samples (automated robotics), to read the reportermolecules (scanners) and analyze the data (bioinformatic tools).Microarray technology can facilitate monitoring of many probes persquare centimeter. Advantages of using multiple probes include, but arenot limited to, speed, adaptability, comprehensiveness and therelatively cheaper cost of high volume manufacturing. The uses of suchan array include, but are not limited to, diagnostic microbiology,including the detection and identification of pathogens, investigationof anti-microbial resistance, epidemiological strain typing,investigation of oncogenes, analysis of microbial infections using hostgenomic expression, and polymorphism profiles.

Recent advances in genomics have culminated in sequencing of entiregenomes of several organisms, including humans. Genomics alone, however,cannot provide a complete understanding of cellular processes that areinvolved in disease, development, and other biological phenomena;because such processes are often directly mediated by polypeptides oftenas participants in ligand-receptor binding reactions. Given the largenumbers of polypeptides are encoded by the genome of an organism, thedevelopment of high throughput technologies for analyzing polypeptidesis of paramount importance.

Peptide arrays with distinct analyte-detecting regions or probes can beassembled on a single substrate by techniques well known to one skilledin the art. A variety of methods are available for creating a peptidemicroarray. These methods include: (a) chemo selective immobilizationmethods; and (b) in situ parallel synthesis methods which can be furtherdivided into (1) SPOT synthesis and (2) photolithographic synthesis.However, chemo selective immobilization methods of the prior art tend tobe cumbersome, requiring multiple steps, or are difficult to controlspatially, limiting the feature density that can be achieved using thesemethods, and in situ parallel synthesis methods of the prior art sufferfrom deficiencies relating to low or inconsistent coupling efficienciesacross multiple coupling cycles. The methods in the prior art sufferfrom slow feature synthesis. The present invention addresses these andother shortcomings of the prior art by providing substrates, systems,and methods for array synthesis and biomolecular analysis as describedin detail below.

SUMMARY

Embodiments of the invention include formulations, substrates, andarrays. Embodiments also include methods for manufacturing and using theformulations, substrates, and arrays. One embodiment includes an arraythat is manufactured using a photoactive coupling formulation, acarboxylic acid activating compound, and a substrate comprisingcarboxylic acid groups. In some embodiments, the photoactive couplingformulation comprises a photoactive compound, a coupling molecule, apolymer, and a solvent. Another embodiment includes an array that ismanufactured using a coupling formulation, a photoactive carboxylic acidactivating compound, and a substrate comprising carboxylic acid groups.In some embodiments, the coupling formulation comprises a couplingmolecule, a polymer, and a solvent. In some embodiments, attaching thecoupling molecule to the substrate comprises selectively exposing eitherthe photoactive compound or the photoactive carboxylic acid activatingcompound to light. In some embodiments, the photoactive compound isabout 0.5-5% by weight of the total formulation.

Examples of coupling molecules include, but are not limited, to aminoacids, peptides, proteins, DNA binding sequences, antibodies,oligonucleotides, nucleic acids, peptide nucleic acids (“PNA”),deoxyribonucleic acids (DNA), ribonucleic acids (RNA), peptide mimetics,nucleotide mimetics, chelates, biomarkers and the like. In oneembodiment, the coupling molecule comprises a naturally occurring orartificial amino acid or polypeptide. In some embodiments, theartificial amino acid is a D-amino acid. In some embodiments, thecoupling molecule is 1-2% by weight of the total formulation. In someembodiments, the coupling molecule comprises a protected group. In someembodiments, the group is protected by Fmoc.

In some embodiments, the photoactive carboxylic acid activating compoundcomprises a carbodiimide precursor compound of formula (I):

wherein

-   -   R is selected from a group comprising hydrogen, substituted or        unsubstituted alkyl, substituted or unsubstituted alkenyl, and        substituted or unsubstituted heterocyclyl, and R further        comprises a water-solubilizing group; and    -   R′ is substituted or unsubstituted alkyl, substituted or        unsubstituted alkenyl, substituted or unsubstituted aryl,        substituted or unsubstituted cycloalkyl, and substituted or        unsubstituted heterocyclyl.

In other embodiments, the photoactive compound comprises a photobasegenerator. Some embodiments comprises a photobase generator compound offormula (II):

wherein

-   -   A^(⊖) is an anion selected from the group consisting of:

-   -    ^(⊖)B(R)₃R′ and ^(⊖)BF₄;    -   R is a substituted or unsubstituted aryl;    -   R′ is an aryl, alkyl, alkenyl, alkoxy, cyano, —NO₂ or fluoro,        said aryl, said alkyl, said alkenyl, and said alkoxy being        optionally substituted;

-   -    is a nitrogen-containing cation, the nitrogen-containing cation        comprising a heteroaryl or heterocyclyl, said heteraryl or        heterocyclyl containing one or more nitrogen atoms.

In some embodiments, the photoactive compound comprises a photobasegenerator compound of formula (II), wherein

-   -   A^(⊖)

-   -    or tetraphenylborate;    -   R″ is hydrogen or —NO₂;

-   -    is

-   -   X is NH or CH₂;    -   n is an integer from 0 to 3; and    -   R′″ is aryl or heteroaryl.

In some embodiments, the photobase generator is1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane or1,3-Bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane. In someembodiments, the photobase generator is carbamate, O-acyloxime, ammoniumsalt, amineimide, α-aminoketone, amidine precursor, or aromatic urea.

Particular embodiments of photobase generator compounds and carbodiimideprecursor compounds are shown in Tables 1-4. More particular embodimentsof photoactive coupling formulations are shown in Table 5.

In certain embodiments, the carboxylic acid activating compound, alsoreferred to as a “coupling reagent” herein, is a carbodiimide. In someembodiments, the coupling reagent is diisopropylcarbodiimide orN-hydroxy-5-norbornene-2,3-dicarboximide. In some embodiments, thepolymer is polymethyl methacrylate.

In some embodiments, the formulations are miscible with water. In someembodiments, the solvent is water, an organic solvent, or a combinationthereof. In certain embodiments, the organic solvent comprises ethyllactate or methylpyrrolidone. In some embodiments, the solvent is about80-90% by weight of the total formulation.

Also encompassed is a substrate, comprising: a first layer, wherein thelayer comprises a plurality of unprotected carboxylic acid groups. Insome embodiments, the first layer is a porous layer. In someembodiments, the carboxylic acid groups are oriented in multipledirections on the surface of the porous layer.

In an embodiment, the first layer is coupled to a support layer. In anembodiment, the first layer is coupled to a silicon wafer. In certainembodiments, the porous layer comprises dextran. In other embodiments,the porous layer comprises porous silica. In an embodiment, the porouslayer comprises pores of a pore size of about 2 nm to 100 μm. In anembodiment, the porous layer comprises a porosity of about 10-80%. In anembodiment, the porous layer comprises a thickness of about 0.01 μm toabout 10,000 μm.

In some embodiments, the substrate further comprises a planar layercomprising a metal having an upper surface and a lower surface. In someembodiments, the first layer is coupled to the planar layer. In someembodiments, the first layer is coated on top of the planar layer. Insome embodiments, the substrate further comprises a plurality of wells.

In an embodiment, the substrate further comprises a plurality of pillarsoperatively coupled to the planar layer in positionally-definedlocations, wherein each pillar has a planar surface extended from theplanar layer, wherein the distance between the surface of each pillarand the upper surface of the layer is between 1,000-5,000 angstroms, andwherein the plurality of pillars are present at a density of greaterthan 10,000/cm², and wherein the first layer is deposited on the planarsurface of the pillars. In some embodiments, the surface area of eachpillar surface is at least 1 μm². In some embodiments, the surface areaof each pillar surface has a total area of less than 10,000 μm². In someembodiments, the distance between the surface of each pillar and thelower surface of the layer is 2,000-7,000 angstroms. In someembodiments, the planar layer is 1,000-2,000 angstroms thick. In someembodiments, the center of each pillar is at least 2,000 angstroms fromthe center of any other pillar. In some embodiments, the surface of eachpillar is parallel to the upper surface of the planar layer. In someembodiments, the surface of each pillar is substantially parallel to theupper surface of the planar layer. In certain embodiments, the metal ischromium. In some embodiments, the metal is chromium, titanium,aluminum, tungsten, gold, silver, tin, lead, thallium, or indium. Insome embodiments, the planar layer is at least 98.5-99% metal by weight.In some embodiments, the planar layer is a homogenous layer of metal. Insome embodiments, each pillar comprises silicon dioxide or siliconnitride. In some embodiments, each pillar is at least 98-99% silicondioxide by weight.

In an embodiment, the substrate further comprises a linker moleculehaving a free amino terminus attached to at least one of the carboxylicacid groups. In some embodiments, the substrate further comprises alinker molecule having a free carboxylic acid group attached to at leastone of the carboxylic acid groups. In some embodiments, the substratefurther comprises a coupling molecule attached to at least one of thecarboxylic acid groups. In some embodiments, the substrate furthercomprises a polymer chain attached to at least one of the carboxylicacid groups.

In an embodiment, the polymer chain comprises a peptide chain. In someembodiments, the polymer chain is attached to at least one of thecarboxylic acid groups via a covalent bond.

Another embodiment encompasses a three-dimensional array of featuresattached to a surface at positionally-defined locations, the featureseach comprising: a collection of peptide chains of determinable sequenceand intended length, wherein within an individual feature, the fractionof peptide chains within the collection having the intended length ischaracterized by an average coupling efficiency for each coupling stepof at least 98%.

In an embodiment, the array comprises a porous layer. In someembodiments, the porous layer comprises a plurality of free carboxylicacid groups. In some embodiments, the porous layer comprises a pluralityof coupling molecules each attached to the array via a carboxylic acidgroup. In some embodiments, the porous layer comprises a plurality ofpeptide chains each attached to the array via a carboxylic acid group.

In certain embodiments, the average coupling efficiency of each couplingstep is at least 98.5%. In some embodiments, the average couplingefficiency of each coupling step is at least 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%. In some embodiments, each peptide chain is from 6 to60 amino acids in length. In some embodiments, each peptide chain is atleast 6 amino acids in length. In some embodiments, each peptide chainis at least 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acidsin length. In some embodiments, each peptide chain comprises one or moreL amino acids. In some embodiments, each peptide chain comprises one ormore D amino acids. In some embodiments, each peptide chain comprisesone or more naturally occurring amino acids. In some embodiments, eachpeptide chain comprises one or more synthetic amino acids. In someembodiments, the array comprises at least 1,000 different peptide chainsattached to the surface. In some embodiments, the array comprises atleast 10,000 different peptide chains attached to the surface.

In an embodiment, each of the positionally-defined locations is at adifferent, known location that is physically separated from each of theother positionally-defined locations. In some embodiments, each of thepositionally-defined locations comprises a plurality of identicalsequences. In some embodiments, each positionally-defined locationcomprises a plurality of identical sequences unique from the otherpositionally-defined locations. In some embodiments, each of thepositionally-defined locations is a positionally-distinguishablelocation. In certain embodiments, each determinable sequence is a knownsequence. In certain embodiments, each determinable sequence is adistinct sequence. In some embodiments, the features are covalentlyattached to the surface. In some embodiments, peptide chains areattached to the surface through a linker molecule or a couplingmolecule.

In certain embodiments, the features comprise a plurality of distinct,nested, overlapping peptide chains comprising subsequences derived froma source protein having a known sequence. In an embodiment, each peptidechain in the plurality is at least 5 amino acids in length. In someembodiments, each peptide chain in the plurality is at least 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length.

In some embodiments, the features comprise a plurality of peptide chainseach having a random, determinable sequence of amino acids.

One embodiment includes a method of attaching a coupling molecule to asubstrate, comprising: obtaining a substrate comprising a plurality ofcarboxylic acid groups for linking to a coupling molecule; contactingthe substrate a carboxylic acid activating compound; contacting thesubstrate with a photoactive coupling formulation comprising aphotoactive compound, a protected coupling molecule, a polymer, and asolvent; selectively exposing the photoactive coupling formulation tolight, thereby deprotecting the protected coupling molecule at aselectively exposed area; coupling the unprotected coupling molecule toat least one of the plurality of carboxylic acid groups at theselectively exposed area; and optionally repeating the method to producea desired polymer at the at least one carboxylic acid group.

Another embodiment includes a method of attaching a coupling molecule toa substrate, comprising: obtaining a substrate comprising a plurality ofcarboxylic acid groups for linking to a coupling molecule; contactingthe substrate with a photoactive carboxylic acid activating compound;selectively exposing the photoactive carboxylic acid activating compoundto light, thereby generating carbodiimide at a selectively exposed areaand activating the carboxylic acid groups on the substrate; contactingthe substrate with a coupling formulation comprising an unprotectedcoupling molecule, a polymer, and a solvent; coupling the unprotectedcoupling molecule to at least one of the plurality of carboxylic acidgroups at the selectively exposed area; and optionally repeating themethod to produce a desired polymer at the at least one carboxylic acidgroup.

In an embodiment, the coupling step has an efficiency of at least 98%.In an embodiment, the coupling molecule is an amino acid. In anembodiment, the polymer is a polypeptide. In an embodiment, thesubstrate comprises a porous layer comprising a plurality of attachmentsites extending in multiple dimensions from the surface of the porouslayer within and around the porous layer. In an embodiment, theattachment site comprises an unprotected carboxylic acid group forbinding to the coupling molecule.

In some embodiments, the substrate comprises a planar layer comprising ametal and having an upper surface and a lower surface; and a pluralityof pillars operatively coupled to the layer in positionally-definedlocations, wherein each pillar has a planar surface extended from thelayer, wherein the distance between the surface of each pillar and theupper surface of the layer is between 1,000-5,000 angstroms, wherein thesurface of each pillar is parallel to the upper surface of the layer,and wherein the plurality of pillars are present at a density of greaterthan 10,000/cm², and wherein the attachment site is coupled to the uppersurface of the pillar.

Another embodiment includes a method of producing a three-dimensionalarray of features, comprising: obtaining a porous layer comprising aplurality of unprotected carboxylic acid groups; and attaching thefeatures to the unprotected carboxylic acid groups, the features eachcomprising a collection of peptide chains of determinable sequence andintended length. In some embodiments, the carboxylic acid groups areoriented in multiple directions.

In some embodiments, within an individual feature, the fraction ofpeptide chains within the collection having the intended length ischaracterized by an average coupling efficiency for each coupling stepof at least 98%. In some embodiments, the features are attached to thesurface using a coupling formulation comprising a solvent, a polymer, acoupling molecule, a neutralization reagent, and a coupling reagent.

One further embodiment includes a method of detecting biomolecules in asample, comprising: providing a substrate comprising at least one porouslayer, wherein the layer comprises a plurality of peptide chainsattached to carboxylic acid groups, wherein the peptide chains have aknown sequence according to positionally-defined locations; contactingthe substrate with the sample; and detecting binding events ofbiomolecules within the sample to the peptide chains. In someembodiments, the carboxylic acid groups are oriented in multipledirections.

In an embodiment, the sample is a biological sample. In an embodiment,the biological sample is a bodily fluid. In some embodiments, the bodilyfluid is amniotic fluid, aqueous humour, vitreous humour, bile, bloodserum, breast milk, cerebrospinal fluid, cerumen, chyle, endolymph,perilymph, feces, female ejaculate, gastric acid, gastric juice, lymph,mucus, peritoneal fluid, pleural fluid, pus, saliva, sebum, semen,sweat, synovial fluid, tears, vaginal secretion, vomit, or urine. Insome embodiments, the biomolecule is a protein. In some embodiments, thebiomolecule is an antibody.

In some embodiments, the method has a greater than 40 fold increase insensitivity of biomolecule detection as compared to a substratecomprising peptide chains attached to a planar layer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, embodiments, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows a measure of density of activated carboxylic acid groups onthe surface of a wafer synthesized by selected methods as describedherein.

FIG. 2 shows a method of manufacturing an array.

FIG. 3A shows the readout of fluorescence signal from each step of thesynthesis of a 20-mer homopolymer.

FIG. 3B shows the coupling efficiency for each addition of a peptide tothe homopolymer.

FIG. 4A shows the readout of fluorescence signal from each step of thesynthesis of a 12-mer heteropolymer.

FIG. 4B shows the coupling efficiency for each addition of a peptide tothe heteropolymer.

FIG. 5 shows a graph of activation lifetimes of carboxylic acids on thesurface of a wafer activated by different activation solvents.

FIG. 6 shows binding of antibodies specific for RHSVV to a peptide arraywith RHSVV and GHSVV sequences synthesized by the methods describedherein.

FIG. 7 shows fluorescein intensity to measure coupling efficiency ofeach amino acid under different experimental conditions as describedherein.

FIG. 8 shows the effect of photobase generator concentration in thephotoresist on coupling efficiency of amino acids to the wafer asdescribed herein.

FIG. 9 shows coupling efficiency of individual coupling after carboxylicacid group activation versus multiple coupling after carboxylic acidgroup activation.

FIG. 10 shows binding of antibodies specific for RHSVV to a peptidearray with RHSVV and GHSVV sequences synthesized by the methodsdescribed herein using a photoacid and a Boc-protected piperidine basein the photoresist composition.

FIG. 11 shows a process flow for developing a protein array usingphotoactivated carbodiimide.

FIG. 12 shows the binding data for TNF alpha and IL-6 protein arrayformed via carbodiimide activation of carboxylic acid bound to thesubstrate.

DETAILED DESCRIPTION

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein the term “wafer” refers to a slice of semiconductormaterial, such as a silicon or a germanium crystal generally used in thefabrication of integrated circuits. Wafers can be in a variety of sizesfrom, e.g., 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimensionwith thickness from, e.g., 275 μm to 775 μm.

As used herein the term “photoresist” or “resist” or “photoactivematerial” refers to a light-sensitive material that changes itssolubility in a solution when exposed to ultra violet or deep ultraviolet radiation. Photoresists are organic or inorganic compounds thatare typically divided into two types: positive resists and negativeresists. A positive resist is a type of photoresist in which the portionof the photoresist that is exposed to light becomes soluble to thephotoresist developer. The portion of the photoresist that is unexposedremains insoluble to the photoresist developer. A negative resist is atype of photoresist in which the portion of the photoresist that isexposed to light becomes insoluble to the photoresist developer. Theunexposed portion of the photoresist is dissolved by the photoresistdeveloper.

As used herein the term “photomask” or “reticle” or “mask” refers to anopaque plate with transparent patterns or holes that allow light to passthrough. In a typical exposing process, the pattern on a photomask istransferred onto a photoresist.

As used herein the term “photoactive compound” refers to compounds thatare modified when exposed to electromagnetic radiation. These compoundsinclude, for example, cationic photoinitiators such as photoacid orphotobase generators, which generate an acid or a base, respectively,when exposed to electromagnetic radiation. A photoinitiator is acompound especially added to a formulation to convert electromagneticradiation into chemical energy in the form of initiating species, e.g.,free radicals or cations. The acid, base, or other product of aphotoactive compound exposed to electromagnetic radiation may then reactwith another compound in a chain reaction to produce a desired chemicalreaction. The spatial orientation of the occurrence of these chemicalreactions is thus defined according to the pattern of electromagneticradiation the solution or surface comprising photoactive compounds isexposed to. This pattern may be defined, e.g., by a photomask orreticle.

As used herein the term “coupling molecule” or “monomer molecule”includes any natural or artificially synthesized amino acid with itsamino group protected with a fluorenylmethyloxycarbonyl (Fmoc or F-Moc)group or a t-butoxycarbonyl (tboc or Boc) group. These amino acids mayhave their side chains protected as an option. Examples of couplingmolecules include Boc-Gly-OH, Fmoc-Trp-OH. Other examples are describedbelow.

As used here in the term “coupling” or “coupling process” or “couplingstep” refers to a process of forming a bond between two or moremolecules such as a linking molecule or a coupling molecule. A bond canbe a covalent bond such as a peptide bond. A peptide bond is a chemicalbond formed between two molecules when the carboxyl group of onecoupling molecule reacts with the amino group of the other couplingmolecule, releasing a molecule of water (H₂O). This is a dehydrationsynthesis reaction (also known as a condensation reaction), and usuallyoccurs between amino acids. The resulting —C(═O)NH— bond is called apeptide bond, and the resulting molecule is an amide.

As used herein the term “coupling efficiency” refers to the probabilityof successful addition of a monomer to a reaction site (e.g., at the endof a polymer) available for binding to the monomer. For example, duringthe growth of a peptide chain in the N to C orientation, a polypeptidehaving a free carboxyl group would bind to an amino acid having a freeamine group under appropriate conditions. The coupling efficiency givesthe probability of the addition of a free amino acid to the freecarboxyl group under certain conditions. It may be determined in bulk,e.g., by monitoring single monomer additions to several unique reactionsites simultaneously.

As used herein the terms “polypeptide,” “peptide,” or “protein” are usedinterchangeably to describe a chain or polymer of amino acids that arelinked together by bonds. Accordingly, the term “peptide” as used hereinincludes a dipeptide, tripeptide, oligopeptide, and polypeptide. Theterm “peptide” is not limited to any particular number of amino acids.In some embodiments, a peptide contains about 2 to about 50 amino acids,about 5 to about 40 amino acids, or about 5 to about 20 amino acids. Amolecule, such as a protein or polypeptide, including an enzyme, can bea “native” or “wild-type” molecule, meaning that it occurs naturally innature; or it may be a “mutant,” “variant,” “derivative,” or“modification,” meaning that it has been made, altered, derived, or isin some way different or changed from a native molecule or from anothermolecule such as a mutant.

As used herein the term “biomarkers” includes, but is not limited toDNA, RNA, proteins (e.g., enzymes such as kinases), peptides, sugars,salts, fats, lipids, ions and the like.

As used herein the term “linker molecule” or “spacer molecule” includesany molecule that does not add any functionality to the resultingpeptide but spaces and extends the peptide out from the substrate, thusincreasing the distance between the substrate surface and the growingpeptide. This generally reduces steric hindrance with the substrate forreactions involving the peptide (including uni-molecular foldingreactions and multi-molecular binding reactions) and so improvesperformance of assays measuring one or more embodiments of peptidefunctionality.

As used herein the term “developer” refers to a solution that canselectively dissolve the materials that are either exposed or notexposed to light. Typically developers are water-based solutions withminute quantities of a base added. Examples include tetramethyl ammoniumhydroxide in water-based developers. Developers are used for the initialpattern definition where a commercial photoresist is used.

As used herein the term “protecting group” includes a group that isintroduced into a molecule by chemical modification of a functionalgroup to obtain chemoselectivity in a subsequent chemical reaction.Chemoselectivity refers to directing a chemical reaction along a desiredpath to obtain a pre-selected product as compared to another. Forexample, the use of tboc as a protecting group enables chemoselectivityfor peptide synthesis using a light mask and a photoacid generator toselectively remove the protecting group and direct predetermined peptidecoupling reactions to occur at locations defined by the light mask.

As used herein the term “microarray,” “array” or “chip” refers to asubstrate on which a plurality of probe molecules of protein or specificDNA binding sequences have been affixed at separate locations in anordered manner thus forming a microscopic array. Protein or specific DNAbinding sequences may be bound to the substrate of the chip through oneor more different types of linker molecules. A “chip array” refers to aplate having a plurality of chips, for example, 24, 96, or 384 chips.

As used herein the term “probe molecules” refers to, but is not limitedto, proteins, DNA binding sequences, antibodies, peptides,oligonucleotides, nucleic acids, peptide nucleic acids (“PNA”),deoxyribonucleic acids (DNA), ribonucleic acids (RNA), peptide mimetics,nucleotide mimetics, chelates, biomarkers and the like. As used herein,the term “feature” refers to a particular probe molecule that has beenattached to a microarray. As used herein, the term “ligand” refers to amolecule, agent, analyte or compound of interest that can bind to one ormore features.

As used herein the term “microarray system” or a “chip array system”refers to a system usually comprised of bio molecular probes formattedon a solid planar surface like glass, plastic or silicon chip plus theinstruments needed to handle samples (automated robotics), to read thereporter molecules (scanners) and analyze the data (bioinformatictools).

As used herein the term “patterned region” or “pattern” or “location”refers to a region on the substrate on which are grown differentfeatures. These patterns can be defined using photomasks.

As used herein the term “derivatization” refers to the process ofchemically modifying a surface to make it suitable for biomolecularsynthesis. Typically derivatization includes the following steps: makingthe substrate hydrophilic, adding an amino silane group, and attaching alinker molecule.

As used herein the term “capping” or “capping process” or “capping step”refers to the addition of a molecule that prevents the further reactionof the molecule to which it is attached. For example, to prevent thefurther formation of a peptide bond, the amino groups are typicallycapped with an acetic anhydride molecule. In other embodiments,ethanolamine is used.

As used herein the term “diffusion” refers to the spread of, e.g.,photoacid or photobase through random motion from regions of higherconcentration to regions of lower concentration.

As used herein the term “dye molecule” refers to a dye which typicallyis a colored substance that can bind to a substrate. Dye molecules canbe useful in detecting binding between a feature on an array and amolecule of interest.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific.

As used herein the term “biological sample” refers to a sample derivedfrom biological tissue or fluid that can be assayed for an analyte(s) ofinterest. Such samples include, but are not limited to, sputum, amnioticfluid, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes. Although the sampleis typically taken from a human patient, the assays can be used todetect analyte(s) of interest in samples from any organism (e.g.,mammal, bacteria, virus, algae, or yeast) or mammal, such as dogs, cats,sheep, cattle, and pigs. The sample may be pretreated as necessary bydilution in an appropriate buffer solution or concentrated, if desired.

As used herein, the term “assay” refers to a type of biochemical testthat measures the presence or concentration of a substance of interestin solutions that can contain a complex mixture of substances.

The term “antigen” as used herein refers to a molecule that triggers animmune response by the immune system of a subject, e.g., the productionof an antibody by the immune system. Antigens can be exogenous,endogenous or auto antigens. Exogenous antigens are those that haveentered the body from outside through inhalation, ingestion orinjection. Endogenous antigens are those that have been generated withinpreviously-normal cells as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. Auto antigens arethose that are normal protein or protein complex present in the hostbody but can stimulate an immune response.

As used herein the term “epitope” or “immunoactive regions” refers todistinct molecular surface features of an antigen capable of being boundby component of the adaptive immune system, e.g., an antibody or T cellreceptor. Antigenic molecules can present several surface features thatcan act as points of interaction for specific antibodies. Any suchdistinct molecular feature can constitute an epitope. Therefore,antigens have the potential to be bound by several distinct antibodies,each of which is specific to a particular epitope.

As used herein the term “antibody” or “immunoglobulin molecule” refersto a molecule naturally secreted by a particular type of cells of theimmune system: B cells. There are five different, naturally occurringisotypes of antibodies, namely: IgA, IgM, IgG, IgD, and IgE.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Unless otherwise noted, “alkyl” as used herein, whether used alone or aspart of a substituent group, refers to a saturated, branched, orstraight-chain monovalent hydrocarbon radical derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane. Typicalalkyl groups include, but are not limited to, methyl; ethyls; propylssuch as propan-1-yl, propan-2-yl; butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl, 2-methyl-propan-2-yl, and the like. In preferredembodiments, the alkyl groups are C₁₋₆alkyl, with C₁₋₃alkyl beingparticularly preferred. “Alkoxyl” radicals are oxygen ethers formed fromthe previously described straight or branched chain alkyl groups.

As used herein, “halo” or “halogen” shall mean chlorine, bromine,fluorine and iodine. “Halo substituted” shall mean a group substitutedwith at least one halogen atom, preferably substituted with a least onefluoro atom. Suitable examples include, but are not limited to —CF₃, andthe like.

The term “cycloalkyl,” as used herein, refers to a stable, saturated orpartially saturated monocyclic or bicyclic ring system containing from 3to 8 ring carbons and preferably 5 to 7 ring carbons. Examples of suchcyclic alkyl rings include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl or cycloheptyl.

The term “alkenyl” refers to an unsaturated branched, straight-chain orcyclic monovalent hydrocarbon radical, which has at least onecarbon-carbon double bond, derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The radical may be ineither the cis or trans conformation about the double bond(s). Typicalalkenyl groups include, but are not limited to, ethenyl; propenyls suchas prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like.

The term “heteroaryl” refers to a monovalent heteroaromatic radicalderived by the removal of one hydrogen atom from a single atom of aparent heteroaromatic ring system. Typical heteroaryl groups includemonocyclic and bicyclic systems where one or both rings isheteroaromatic. Heteroaromatic rings may contain 1-4 heteroatomsselected from O, N, and S. Examples include but are not limited to,radicals derived from carbazole, furan, imidazole, indazole, indole,indolizine, isoindole, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, purine, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

The term “aryl,” as used herein, refers to aromatic groups comprising astable six-membered monocyclic, or ten-membered bicyclic orfourteen-membered tricyclic aromatic ring system which consists ofcarbon atoms. Examples of aryl groups include, but are not limited to,phenyl or naphthalenyl.

The term “heterocyclyl” is a 3- to 12-member saturated or partiallysaturated single (monocyclic), bicyclic, or fused ring system whichconsists of carbon atoms and from 1 to 6 heteroatoms selected from N, Oand S. The heterocyclyl group may be attached at any heteroatom orcarbon atom which results in the creation of a stable structure. Thebicyclic heterocyclyl group includes systems where one or both ringsinclude heteroatoms. Examples of heterocyclyl groups include, but arenot limited to, 2-imidazoline, imidazolidine; morpholine, oxazoline,oxazolidine, 2-pyrroline, 3-pyrroline, pyrrolidine, pyridone,pyrimidone, piperazine, piperidine, indoline, tetrahydrofuran,2-pyrroline, 3-pyrroline, 2-imidazoline, 2-pyrazoline, indolinone,thiomorpholine, tetrahydropyran, tetrahydroquinoline,tetrahydroquinazoline, [1,2,5]thiadiazolidine 1,1-dioxide,[1,2,3]oxathiazolidine 2,2-dioxide, and the like.

The term “cis-trans isomer” refers to stereoisomeric olefins orcycloalkanes (or hetero-analogues) which differ in the positions ofatoms (or groups) relative to a reference plane: in the cis-isomer theatoms of highest priority are on the same side; in the trans-isomer theyare on opposite sides.

The term “substituted” refers to a radical in which one or more hydrogenatoms are each independently replaced with the same or differentsubstituent(s).

With reference to substituents, the term “independently” means that whenmore than one of such substituent is possible, such substituents may bethe same or different from each other.

The term “oxo” whether used alone or as part of a substituent grouprefers to an O=bounded to either a carbon or a sulfur atom. For example,phthalimide and saccharin are examples of compounds with oxosubstituents.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

Compounds

In some embodiments, the photoactive carboxylic acid activating compoundcomprises a carbodiimide precursor compound of formula (I):

wherein

-   -   R is selected from a group comprising hydrogen, substituted or        unsubstituted alkyl, substituted or unsubstituted alkenyl, and        substituted or unsubstituted heterocyclyl, and R further        comprises a water-solubilizing group; and    -   R′ is substituted or unsubstituted alkyl, substituted or        unsubstituted alkenyl, substituted or unsubstituted aryl,        substituted or unsubstituted cycloalkyl, and substituted or        unsubstituted heterocyclyl.

In other embodiments, the photoactive compound comprises a photobasegenerator. Some embodiments comprises a photobase generator compound offormula (II):

wherein

-   -   A^(⊖) is an anion selected from the group consisting of:

-   -    ^(⊖)B(R)₃R′ and ^(⊖)BF₄;    -   R is a substituted or unsubstituted aryl;    -   R′ is an aryl, alkyl, alkenyl, alkoxy, cyano, —NO₂ or fluoro,        said aryl, said alkyl, said alkenyl, and said alkoxy being        optionally substituted;

-   -    is a nitrogen-containing cation, the nitrogen-containing cation        comprising a heteroaryl or heterocyclyl, said heteraryl or        heterocyclyl containing one or more nitrogen atoms.

In some embodiments, the photoactive compound comprises a photobasegenerator compound of formula (II), wherein

-   -   A^(⊖) is

-   -    or tetraphenylborate;    -   R′″ is hydrogen or —NO₂;

-   -    is

-   -   X is NH or CH₂;    -   n is an integer from 0 to 3; and    -   R′″ is aryl or heteroaryl.

In some embodiments, the photobase generator is carbamate, O-acyloxime,ammonium salt, amineimide, α-aminoketone, amidine precursor, or aromaticurea.

In certain embodiments, the carboxylic acid activating compound, alsoreferred to as a “coupling reagent” herein, is a carbodiimide. In someembodiments, the coupling reagent is diisopropylcarbodiimide orN-hydroxy-5-norbornene-2,3-dicarboximide. In some embodiments, thepolymer is polymethyl methacrylate.

Representative photoactive carboxylic acid activating compound relatedto the present invention are listed in Table 1 below:

TABLE 1 Carbodiimide Precursor Compounds

Compound # R R′ Name 1

1-(hydroxymethyl)-4-phenyl- 1,4-dihydro-5H-tetrazole-5- thione 2

1,4-Bis(2,2-dimethyl-1,3- dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione 3

1-(3-(dimethylamino)propyl)- 4-ethyl-1,4-dihydro-5H- tetrazole-5-thione4 H

1-cyclohexyl-1,4-dihydro-5H- tetrazole-5-thione 5

1-phenyl-4-(piperidin-1- ylmethyl)-1,4-dihydro-5H- tetrazole-5-thione 6

1-(3-(diethylamino)propyl)-4- (2-methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione 7

1-((diethylamino)methyl)-4- phenyl-1,4-dihydro-5H- tetrazole-5-thione

Representative photoactive compounds related to the present inventionare listed in Table 2 below:

TABLE 2 Nonionic Photobase Generator Compounds Compound # Structure Name8

1,3-Bis[(2- nitrobenzyl)oxycarbonyl-4- piperidyl]propane 9

1,3-Bis[1-(9- fluorenylmethoxycarbonyl)- 4-piperidyl]propane

Representative photoactive compounds related to the present inventionare listed in Table 3 below:

TABLE 3 Ionic Photobase Generator Compounds

      Compound #         R

        Name 10 H

1,5,7- triazabicyclo[4.4.0]dec-5- enyl-phenylglyoxylate 11 NO₂

1,5,7- triazabicyclo[4.4.0]dec-5- enyl-4- nitrophenylglyoxylate

      Compound #

        Name 12

1,5,7- triazabicyclo[4.4.0]dec-5- enyl-tetraphenylborate 13

1,8- Diazabicyclo[5.4.0]undec- 7-enyl-tetraphenylborate 14

1-Phenacyl-(1-azonia-4- azabicyclo[2,2,2]octane)- tetraphenylborate 15

1-Naphthoylmethyl-(1- azonia-4- azabicyclo[2,2,2]octane)-tetraphenylborate

Photoactive carboxylic acid activating compound related to the presentinvention are listed in Table 4 below:

TABLE 4 Carbodiimide Precursor Compounds

Compound # R R′ Name 16 

1-(3-(diethylamino)propyl)-4- phenyl-1,4-dihydro-5H- tetrazole-5-thione17 

1-(3-(diethylamino)propyl)-4- (methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione 18 

1-(3-(diethylamino)propyl)-4- (dimethylamino-phenyl)-1,4-dihydro-5H-tetrazole-5-thione 19 

1-(3-(diethylamino)propyl)-4- (methylthio-phenyl)-1,4-dihydro-5H-tetrazole-5-thione 20 

1-(3-(diethylamino)propyl)-4- (nitrophenyl)-1,4-dihydro-5H-tetrazole-5-thione 21 

1-(3-(diethylamino)propyl)-4- (ethoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione 22 

1-([1,1′-biphenyl]-4-yl)-4-(3- (diethylamino)propyl)-1,4-dihydro-5H-tetrazole-5-thione 23 

1-(3-(diethylamino)propyl)-4- (4-methoxynaphthalen-1-yl)-1,4-dihydro-5H-tetrazole-5- thione 24¹

alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each substitutedor unsubstituted 25  alkyl, alkenyl and alkyl, alkenyl, heterocyclyl,each cycloalkyl, heterocyclyl substituted or and aryl, e.g.unsubstituted and phenyl, biphenyl, comprising a water naphthyl, eachsolubilizing group substituted or unsubstituted ¹commercially available

Synthesis

This application provides methods of making the disclosed compoundsaccording to traditional organic synthetic methods as well as matrix orcombinatorial synthetic methods. Scheme 1 describe suggested syntheticroutes. Using the scheme, the guidelines below, and the examples, aperson of skill in the art may develop analogous or similar methods fora given compound that is within the invention. These methods arerepresentative of the synthetic schemes, but are not to be construed aslimiting the scope of the invention.

Where the compounds according to this invention have at least one chiralcenter, they may accordingly exist as enantiomers. Where the compoundspossess two or more chiral centers, they may additionally exist asdiastereomers. Where the processes for the preparation of the compoundsaccording to the invention give rise to mixtures of stereoisomers, theseisomers may be separated by techniques such as preparativechromatography. The compounds may be prepared in racemic form or asindividual enantiomers or diasteromers by either stereospecificsynthesis or by resolution. The compounds may, for example, be resolvedinto their component enantiomers or diastereomers by techniques, such asthe formation of stereoisomeric pairs by salt formation with anoptically active base, followed by fractional crystallization andregeneration of the free acid. The compounds may also be resolved byformation of stereoisomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, the compounds may be resolved using a chiral HPLC column.It is to be understood that all stereoisomers, racemic mixtures,diastereomers, geometric isomers, and enantiomers thereof areencompassed within the scope of the present invention.

Furthermore, some of the crystalline forms for the compounds may existas polymorphs and as such are intended to be included in the presentinvention. In addition, some of the compounds may form solvates withwater (i.e., hydrates) or common organic solvents, and such solvates arealso intended to be encompassed within the scope of this invention.

Examples of the described synthetic routes include Scheme 1 and Examples1 through 12. Compounds analogous to the target compounds of theseexamples can be made according to similar routes. The disclosedcompounds are useful in the manufacture of microarrays as describedherein.

General Guidance

The compound (I), wherein R is hydroxymethyl,2,2-dimethyl-1,3-dioxolan-4-ylmethyl, piperidin-1-ylmethyl, hydrogen,2,2-dimethyl-1,3-dioxolan-4-yl, piperidin-1-ylmethyl,3-(diethylamino)propyl, alkyl, aryl, heterocyclyl, cycloalkyl or asdefined in the above formula (I), can be synthesized as outlined by thegeneral synthetic route illustrated in Scheme 1. Treatment of anappropriate isothiocyanate (I) with sodium azide, an known compoundprepared by known methods in water solution of isopropanol at 80°Celsius for 3 hours yields the R-substituted1-hydro-5H-tetrazole-5-thione (II) following a 1,3 dipolarcycloaddition. Copper mediated cross-coupling of the R-substituted1-hydro-5H-tetrazole-5-thione (II) with phenylboronic acid (III) in thepresence of copper acetate, pyridine and dimethylformamide (DMF) at atemperature of 60° Celsius for 18 hours yields compound (IV).

Scheme 1 provides a 70% yield of compound (II) and 33% yield of compound(IV) when R is diethylaminopropyl. The amine of compound (IV) isprotonated in the presence of hydrochloride in methanol according to:

Scheme 2 provides the general scheme of photoactivated carbodiimideformation, e.g. photoinduced formation ofhydroxymethyl-phenyl-carbodiimide or1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-carbodiimide.

Scheme 3 provides the general scheme of photoactivated base formation,e.g. 1,3-di(piperidin-4-yl)propane, from a non-ionic photobase generatorcompounds.

Formulations

Disclosed herein are formulations such as photoactive couplingformulations and linker formulations. These formulations can be usefulin the manufacture and/or use of, e.g., substrates and/or peptide arraysdisclosed herein. Generally the components of each formulation disclosedherein are soluble in water at room temperature (app. 25° C.).

Photoactive Coupling Formulations

Disclosed herein are photoactive coupling formulations. In someembodiments, a photoactive coupling formulation can include componentssuch as a solvent, a coupling reagent, a coupling molecule, aphotoactive compound, and a polymer. In some embodiments, photoactivecoupling formulations are shown in Table 5.

In one aspect, a photoactive coupling formulation can include aphotoactive compound. Photoactive compounds may include photobase orphotoacid generators. Exposure of the photoactive compounds toelectromagnetic radiation is a primary photochemical event that producesa compound that goes on to induce material transforming secondaryreactions within a diffusion-limited radius. A photoactive couplingformulation may comprise a photoactive compound comprising aradiation-sensitive catalyst precursor, e.g., a photoacid generator(PAG); a plurality of chemical groups that can react by elimination,addition, or rearrangement in the presence of catalyst; and optionaladditives to improve performance or processability, e.g., surfactants,photosensitizers, and etch resistors.

In some embodiments, a photoactive coupling formulation includes aphotobase generator and a photo sensitizer in a polymer matrix dispersedin a solvent. In some embodiments, the polymer in the composition of thephotoresist is generally inert and non-crosslinking but the photoactivecompounds will readily generate sufficient quantities of photobase uponexposure to electromagnetic radiation to bring about a desired reactionto produce a product at acceptable yield.

In some embodiments, a photoactive coupling formulation can includevarious components such as a photosensitizer, a photoactive compound, apolymer, and a solvent. Specific examples of photoactive couplingformulations are shown in Table 5.

In some embodiments, a photoactive compound can be a photoacid generator(PAG) or a photobase generator (PBG). Photoacid generators (or PAGs) arecationic photoinitiators. A photoinitiator is a compound especiallyadded to a formulation to convert absorbed light energy, UV or visiblelight, into chemical energy in the form of initiating species, e.g.,free radicals or cations. Cationic photoinitiators are used extensivelyin optical lithography. The ability of some types of cationic photoinitiators to serve as latent photochemical sources of very strongprotonic or Lewis acids is generally the basis for their use in photoimaging applications. In some embodiments, a photoacid generator is aniodonium salt, a polonium salt, or a sulfonium salt. In someembodiments, a photoacid generator is (4-Methoxyphenyl)phenyliodonium ortrifluoromethanesulfonate. In some embodiments, a photoacid generator is(2,4-dihydroxyphenyl)dimethylsulfonium triflate or (4methoxyphenyl)dimethylsulfonium triflate, shown below:

In some embodiments, a photoacid generator is iodonium and sulfoniumsalts of triflates, phosphates and/or antimonates. In some embodiments,a photoacid generator is about 0.5-5% by weight of the total formulationconcentration. In some embodiments, a photoacid generator is about lessthan 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0%by weight of the total formulation concentration.

In some embodiments, a photobase generator is1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane or1,3-Bis[(1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane. Thephotobase generator should be present in a composition of the inventionin an amount sufficient to enable deprotection of the monomer so thatthey are available for binding to the substrate. In some embodiments, aphotobase generator is about 0.5-5% by weight of the total formulationconcentration. In some embodiments, a photobase generator is about lessthan 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0%by weight of the total formulation concentration.

In some embodiments, a polymer is a non-crosslinking inert polymer. Insome embodiments, a polymer is a polyvinyl pyrrolidone. The generalstructure of polyvinyl pyrrolidone is as follows, where n is anypositive integer greater than 1:

In some embodiments, a polymer is a polymer of vinyl pyrrolidone. Insome embodiments, a polymer is polyvinyl pyrrolidone. Poly vinylpyrrollidone is soluble in water and other polar solvents. When dry itis a light flaky powder, which generally readily absorbs up to 40% ofits weight in atmospheric water. In solution, it has excellent wettingproperties and readily forms films. In some embodiments, a polymer is avinyl pyrrolidone or a vinyl alcohol. In some embodiments, a polymer isa polymethyl methacrylate.

In some embodiments, a polymer is 2.5-5% by weight of the totalformulation concentration. In some embodiments, a polymer is about0.5-5% by weight of the total formulation concentration. In someembodiments, a polymer is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, or greater than 5.0% by weight of the total formulationconcentration.

In some embodiments, a solvent is water, ethyl lactate, n methylpyrrollidone or a combination thereof. In some embodiments, ethyllactate can be dissolved in water to more than 50% to form a solvent. Insome embodiments, a solvent can be about 10% propylene glycol methylether acetate (PGMEA) and about 90% DI water. In some embodiments, asolvent can include up to about 20% PGMEA. In some embodiments, asolvent can include 50% ethyl lactate and 50% n methyl pyrrollidone. Insome embodiments, a solvent is n methyl pyrrollidone. In someembodiments, a solvent is water, an organic solvent, or combinationthereof. In some embodiments, the organic solvent is N Methylpyrrolidone, di methyl formamide or combinations thereof.

In some embodiments, the solvent is about 80-90% by weight of the totalformulation concentration. In some embodiments, the solvent is aboutless than 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, orgreater than 99% by weight of the total formulation concentration.

The photoactive coupling formulation comprises coupling molecules. Thecoupling molecules can include amino acids. In some instances allpeptides on an array described herein are composed of naturallyoccurring amino acids. In others, peptides on an array described hereincan be composed of a combination of naturally occurring amino acids andnon-naturally occurring amino acids. In other cases, peptides on anarray can be composed solely from non-naturally occurring amino acids.Non-naturally occurring amino acids include peptidomimetics as well asD-amino acids. The R group can be found on a natural amino acid or agroup that is similar in size to a natural amino acid R group.Additionally, unnatural amino acids, such as beta-alanine,phenylglycine, homoarginine, aminobutyric acid, aminohexanoic acid,aminoisobutyric acid, butylglycine, citrulline, cyclohexylalanine,diaminopropionic acid, hydroxyproline, norleucine, norvaline, ornithine,penicillamine, pyroglutamic acid, sarcosine, and thienylalanine can alsobe incorporated. These and other natural and unnatural amino acids areavailable from, for example, EMD Biosciences, Inc., San Diego, Calif. Insome embodiments, a coupling molecule comprises a naturally occurring orartificial amino acid or polypeptide. Examples of coupling moleculesinclude Boc-Glycine-OH and Boc-Histidine-OH. In some embodiments, theartificial amino acid is a D-amino acid. In some embodiments, a couplingmolecule is 1-2% by weight of the total formulation concentration. Insome embodiments, a coupling molecule is about 0.5-5% by weight of thetotal formulation concentration. In some embodiments, a couplingmolecule is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0% by weight of the total formulation concentration. Insome embodiments, a coupling molecule comprises a protected group, e.g.,a group protected via t-Boc or F-Moc chemistry. In most instances,increasing the concentration of a coupling molecule provides the bestperformance.

In some embodiments, a coupling reagent is carbodiimide or triazole. Insome embodiments, a coupling reagent is N-Hydroxysuccinimide (NHS). Insome embodiments, a coupling reagent is 2-4% by weight of the totalformulation concentration. In some embodiments, a coupling reagent isabout 0.5-5% by weight of the total formulation concentration. In someembodiments, a coupling reagent is about less than 0.1, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% by weight of the totalformulation concentration.

In any of the combinations above, the formulation can be completelywater strippable. Thus, in some embodiments, water can be used to washaway the photoactive coupling formulation after exposure.

Carboxylic Acid Activating Formulations

Disclosed herein are activating formulations for activating carboxylicacid so that it reacts with a free amino group of a biomolecule, e.g.,an amino acid, peptide, or polypeptide. An activating formulation caninclude components such as a carboxylic acid group activating compoundand a solvent. In some embodiments, the carboxylic acid group activatingcompound is a carbodiimide or a carbodiimide precursor. In someembodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the carboxylic acid group activatingcompound is N-Hydroxysuccinimide (NHS). In some embodiments, thecarboxylic acid group activating compound is selected from:1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC],N-hydroxysuccinimide [NHS], 1,3-Diisopropylcarbodiimide [DIC],hydroxybenzotriazole (HOBt),(0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) [HATU],benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP], and N,N-Diisopropylethylamine [DIEA]. In some embodiments, thesolvent is water. In some embodiments, the solvent isN-methylpyrrolidone (NMP). In some embodiments, the carboxylic acidgroup activating compound converts the carboxylic acid to a carbonylgroup (i.e., carboxylic acid group activation). In some embodiments, thecarboxylic acid group is activated for 5, 10, 15, 20, 30, 45, or 60minutes after exposure to an activation formulation.

In some embodiments, the activating formulation comprises 4% by weightof 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 2% by weight ofN-hydroxysuccinimide (NHS) dissolved in deionized water. In someembodiments, the activating formulation comprises 4% by weight of1,3-Diisopropylcarbodiimide (DIC) and 2% by weight ofhydroxybenzotriazole (HOBt) dissolved in NMP. In some embodiments, theactivating formulation comprises 4% by weight of(0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) (HATU) and 2% by weight ofN,N-Diisopropylethylamine (DIEA) dissolved in NMP. In some embodiments,the activating formulation comprises 4% by weight ofBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) and 2% by weight of DIEA dissolved in NMP.

In some embodiments, the carboxylic acid group activating compound is acarbodiimide precursor. In one aspect, the carbodiimide precursor isconverted to a carbodiimide through exposure to radiation, e.g.,ultraviolet radiation. In one embodiment, the carbodiimide precursor isa thione. The carbodiimide precursor can also be referred to as aphotoactivated carbodiimide. In one embodiment, photoactivatedcarbodiimides are used to provide site-specific activation of carboxylicacid groups on an array by spatially controlling exposure of thephotoactivated carbodiimide solution to electromagnetic radiation at apreferred activation wavelength. In some embodiments, the preferredactivation wavelength is 248 nm.

In one embodiment, the carbodiimide precursor is a thione that isconverted to carbodiimide via photoactivation. In one aspect, the thioneis converted to a hydroxymethyl phenyl carbodiimide after exposure toelectromagnetic radiation. In some embodiments, the thione is4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione,4-cyclohexyl-1H-tetrazole-5(4H)-thione, or 1-phenyl-4-(piperidinomethyl)tetrazole-5(4H)-thione, and others as shown in Tables 1 and 4.

In some embodiments, the activating solution comprises a carbodiimideprecursor, a solvent, and a polymer. In one embodiment, the carbodiimideprecursor is4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,or1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione.In some embodiments, the carbodiimide precursor is present in theactivation solution at a concentration of 2.5% by weight. In someembodiments the carbodiimide precursor is present in the activationsolution at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or5.0% by weight of the total formulation concentration.

In some embodiments, the solvent is water. In some embodiments, thesolvent is about 80-90% by weight of the total formulationconcentration. In some embodiments, the solvent is about less than 70,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or greater than 99% byweight of the total formulation concentration.

In some embodiments, a polymer is a polyvinyl pyrrolidone and/or apolyvinyl alcohol. In some embodiments, a polymer is about 0.5-5% byweight of the total formulation concentration. In some embodiments, apolymer is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0% by weight of the total formulation concentration.

In some embodiments, a coupling reagent is a carbodiimide. In someembodiments, a coupling reagent is a triazole. In some embodiments, acoupling reagent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Insome embodiments, a coupling reagent is about 0.5-5% by weight of thetotal formulation concentration. In some embodiments, a coupling reagentis about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0% by weight of the total formulation concentration.

Linker Formulations

Also disclosed herein is a linker formulation. A linker formulation caninclude components such as a solvent, a polymer, a linker molecule, anda coupling reagent. In some embodiments, the polymer is 1% by weightpolyvinyl alcohol and 2.5% by weight poly vinyl pyrrollidone, the linkermolecule is 1.25% by weight polyethylene oxide, the coupling reagent is1% by weight 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and thesolvent includes water. In some embodiments, the polymer is 0.5-5% byweight polyvinyl alcohol and 0.5-5% by weight poly vinyl pyrrollidone,the linker molecule is 0.5-5% by weight polyethylene oxide, the couplingreagent is 0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water.

In some embodiments, the solvent is water, an organic solvent, or acombination thereof. In some embodiments, the organic solvent is Nmethyl pyrrolidone, dimethyl formamide, dichloromethane, dimethylsulfoxide, or a combination thereof. In some embodiments, the solvent isabout 80-90% by weight of the total formulation concentration. In someembodiments, the solvent is about less than 70, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or greater than 99% by weight of the totalformulation concentration.

In some embodiments, a polymer is a polyvinyl pyrrolidone and/or apolyvinyl alcohol. The general structure of polyvinyl alcohol is asfollows, where n is any positive integer greater than 1:

In some embodiments, a polymer is about 0.5-5% by weight of the totalformulation concentration. In some embodiments, a polymer is about lessthan 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0%by weight of the total formulation concentration.

A linker molecule can be a molecule inserted between a surface disclosedherein and peptide that is being synthesized via a coupling molecule. Alinker molecule does not necessarily convey functionality to theresulting peptide, such as molecular recognition functionality, but caninstead elongate the distance between the surface and the peptide toenhance the exposure of the peptide's functionality region(s) on thesurface. In some embodiments, a linker can be about 4 to about 40 atomslong to provide exposure. The linker molecules can be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units(PEGs), diamines, diacids, amino acids, and combinations thereof.Examples of diamines include ethylene diamine and diamino propane.Alternatively, linkers can be the same molecule type as that beingsynthesized (e.g., nascent polymers or various coupling molecules), suchas polypeptides and polymers of amino acid derivatives such as forexample, amino hexanoic acids. In some embodiments, a linker molecule isa molecule having a carboxylic group at a first end of the molecule anda protecting group at a second end of the molecule. In some embodiments,the protecting group is a t-Boc protecting group or an Fmoc protectinggroup. In some embodiments, a linker molecule is or includes an arylacetylene, a polyethyleneglycol, a nascent polypeptide, a diamine, adiacid, a peptide, or combinations thereof. In some embodiments, alinker molecule is about 0.5-5% by weight of the total formulationconcentration. In some embodiments, a linker molecule is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

The unbound (or free end) portion of a linker molecule can have areactive functional group which is blocked, protected, or otherwise madeunavailable for reaction by a removable protecting group. The protectinggroup can be bound to a linker molecule to protect a reactivefunctionality on the linker molecule. Protecting groups that can be usedinclude all acid- and base-labile protecting groups. For example, linkeramine groups can be protected by t-butoxycarbonyl (t-BOC or BOC) orbenzyloxycarbonyl (CBZ), both of which are acid labile, or by9-fluorenylmethoxycarbonyl (FMOC), which is base labile.

Additional protecting groups that can be used include acid-labile groupsfor protecting amino moieties: tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha,alpha-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9 fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid-labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl. See also, Greene, T. W., Protective Groups in OrganicSynthesis, Wiley-Interscience, NY, (1981).

Photobase Generator Compositions

Disclosed herein are photobase generator compositions. Photobasegenerator compositions can be used to deprotect an Fmoc protected aminoacid upon exposure to light, e.g., through a reticle. In someembodiments, the photobase generator comprises an amine. In someembodiments, the anion is a borate. In some embodiments, the anion is aphenylglyoxylate. In some embodiments, the amine has the formula NR₁R₂R₃with R₁, R₂ and R₃ defined above in formula (II). In some embodiments,the photobase generator comprises an amine attached to a polymer. Insome embodiments, the amine is bound to a counter ion. In oneembodiment, the counter ion is a carboxylate. In one aspect, thecarboxylate undergoes photodecarboxylation upon exposure to radiation.In some embodiments, the counter ion is a borate. In some embodiments,the anion is a phenylglyoxylate. In some embodiments, the photobasegenerator comprises a chromophore attached to an amine and an anion. Insome embodiments, the anion is a borate. In some embodiments, the anionis a phenylglyoxylate.

Also disclosed herein are photobase generator compositions comprising aphotobase generator, a polymer, and an amino acid. In some embodiments,the amino acid is an Fmoc-protected amino acid. In some embodiments, theamino acid is present at 0.1 M in said photobase generator composition.In some embodiments, the polymer is present at 0.5-3% by weight in saidphotobase generator composition. In some embodiments, the polymer ispolymethyl methacrylate.

Substrates

Also disclosed herein are substrates. In some embodiments a substratesurface is planar (i.e., 2-dimensional). In some embodiments a substratesurface is functionalized with free carboxylic acid groups. In someembodiments, a substrate surface is functionalized with free aminegroups. A surface that is functionalized with free amine groups can beconverted to free carboxylic acid groups by reacting with activating thecarboxylic acid groups of a molecule comprising at least two freecarboxylic acid groups (e.g., converting the carboxylic acid group to acarbonyl group using carbodiimide) and reacting the molecule with thefree amine groups attached to the surface of the substrate. In someembodiments, the molecule comprising multiple carboxylic acid groups issuccinic anhydride, polyethylene glycol diacid,benzene-1,3,5-tricarboxylic acid, benzenehexacarboxylic acid, orcarboxymethyl dextran.

In some embodiments, a substrate can include a porous layer (i.e., a3-dimensional layer) comprising functional groups for binding a firstmonomer building block. In some embodiments, a substrate surfacecomprises pillars for peptide attachment or synthesis. In someembodiments, a porous layer is added to the top of the pillars.

Porous Layer Substrates

Porous layers that can be used are flat, permeable, polymeric materialsof porous structure that have a carboxylic acid functional group (thatis native to the constituent polymer or that is introduced to the porouslayer) for attachment of the first peptide building block. For example,a porous layer can be comprised of porous silicon with functional groupsfor attachment of a polymer building block attached to the surface ofthe porous silicon. In another example, a porous layer can comprise across-linked polymeric material. In some embodiments, the porous layercan employ polystyrenes, saccharose, dextrans, polyacryloylmorpholine,polyacrylates, polymethylacrylates, polyacrylamides,polyacrylolpyrrolidone, polyvinylacetates, polyethyleneglycol, agaroses,sepharose, other conventional chromatography type materials andderivatives and mixtures thereof. In some embodiments, the porous layerbuilding material is selected from: poly(vinyl alcohol), dextran, sodiumalginate, poly(aspartic acid), poly(ethylene glycol), poly(ethyleneoxide), poly(vinyl pyrrolidone), poly(acrylic acid), poly(acrylicacid)-sodium salt, poly(acrylamide), poly(N-isopropyl acrylamide),poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(2-acrylamido-2-methyl-1-propanesulfonic acid),polysaccharides, and cellulose derivatives. Preferably the porous layerhas a porosity of 10-80%. In one embodiment, the thickness of the porouslayer ranges from 0.01 μm to about 1,000 μm. Pore sizes included in theporous layer may range from 2 nm to about 100 μm.

According to another embodiment of the present invention there isprovided a substrate comprising a porous polymeric material having aporosity from 10-80%, wherein reactive groups are chemically bound tothe pore surfaces and are adapted in use to interact, e.g. by bindingchemically, with a reactive species, e.g., deprotected monomericbuilding blocks or polymeric chains. In one embodiment the reactivegroup is a carboxylic acid group. The carboxylic acid group is free tobind, for example, an unprotected amine group of a peptide orpolypeptide.

In an embodiment, the porous layer is in contact with a support layer.The support layer comprises, for example, metal, plastic, silicon,silicon oxide, or silicon nitride. In another embodiment, the porouslayer can be in contact with a patterned surface, such as on top ofpillar substrates described below.

Pillar Substrates

In some embodiments, a substrate can include a planar layer comprising ametal and having an upper surface and a lower surface; and a pluralityof pillars operatively coupled to the layer in positionally-definedlocations, wherein each pillar has a planar surface extended from thelayer, wherein the distance between the surface of each pillar and theupper surface of the layer is between about 1,000-5,000 angstroms, andwherein the plurality of pillars are present at a density of greaterthan about 10,000/cm².

In some embodiments, the distance between the surface of each pillar andthe upper surface of the later can be between about less than 1,000,2,000, 3,000, 3,500, 4,500, 5,000, or greater than 5,000 angstroms (orany integer in between).

In some embodiments, the surface of each pillar is parallel to the uppersurface of the layer. In some embodiments, the surface of each pillar issubstantially parallel to the upper surface of the layer.

In some embodiments, the plurality of pillars are present at a densityof greater than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, or 12,000/cm² (or any integer in between).In some embodiments, the plurality of pillars are present at a densityof greater than 10,000/cm². In some embodiments, the plurality ofpillars are present at a density of about 10,000/cm² to about 2.5million/cm² (or any integer in between). In some embodiments, theplurality of pillars are present at a density of greater than 2.5million/cm².

In some embodiments, the surface area of each pillar surface is at least1 μm². In some embodiments, the surface area of each pillar surface canbe at least 0.1, 0.5, 12, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, or 50 μm² (or any integer in between). In some embodiments, thesurface area of each pillar surface has a total area of less than 10,000μm². In some embodiments, the surface area of each pillar surface has atotal area of less than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 μm² (or any integer inbetween).

In some embodiments, the distance between the surface of each pillar andthe lower surface of the layer is 2,000-7,000 angstroms. In someembodiments, the distance between the surface of each pillar and thelower surface of the layer is about less than 500, 1,000, 2,000, 3,000,4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, orgreater than 12,000 angstroms (or any integer in between). In someembodiments, the distance between the surface of each pillar and thelower surface of the layer is 7,000, 3,000, 4,000, 5,000, 6,000, or7,000 angstroms (or any integer in between).

In some embodiments, the layer is 1,000-2,000 angstroms thick. In someembodiments, the layer is about less than 500, 1,000, 2,000, 3,000,4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, orgreater than 12,000 angstroms thick (or any integer in between).

In some embodiments, the center of each pillar is at least 2,000angstroms from the center of any other pillar. In some embodiments, thecenter of each pillar is at least about 500, 1,000, 2,000, 3,000, or4,000 angstroms (or any integer in between) from the center of any otherpillar. In some embodiments, the center of each pillar is at least about2 μm to 200 μm from the center of any other pillar.

In some embodiments, the metal is chromium. In some embodiments, themetal is chromium, titanium, aluminum, tungsten, gold, silver, tin,lead, thallium, indium, or a combination thereof. In some embodiments,the layer is at least 98.5-99% (by weight) metal. In some embodiments,the layer is 100% metal. In some embodiments, the layer is at leastabout greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, or 99%metal. In some embodiments, the layer is a homogenous layer of metal.

In some embodiments, at least one or each pillar comprises silicon. Insome embodiments, at least one or each pillar comprises silicon dioxideor silicon nitride. In some embodiments, at least one or each pillar isat least 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, or 99% (by weight)silicon dioxide.

In some embodiments, a substrate can include a linker molecule having afree amino terminus attached to the surface of each pillar. In someembodiments, a substrate can include a linker molecule having a freeamino terminus attached to the surface of at least one pillar. In someembodiments, a substrate can include a linker molecule having aprotecting group attached to the surface of each pillar. In someembodiments, a substrate can include a linker molecule having aprotecting group attached to the surface of at least one pillar. In someembodiments, a substrate can include a coupling molecule attached to thesurface of at least one pillar. In some embodiments, a substrate caninclude a coupling molecule attached to the surface of each pillar. Insome embodiments, a substrate can include a polymer in contact with thesurface of at least one of the pillars. In some embodiments, a substratecan include a polymer in contact with the surface of each pillar. Insome embodiments, a substrate can include a gelatinous form of a polymerin contact with the surface of at least one of the pillars. In someembodiments, a substrate can include a solid form of a polymer incontact with the surface of at least one of the pillars.

In some embodiments, the surface of at least one of the pillars of thesubstrate is derivatized. In some embodiments, a substrate can include apolymer chain attached to the surface of at least one of the pillars. Insome embodiments, the polymer chain comprises a peptide chain. In someembodiments, the attachment to the surface of the at least one pillar isvia a covalent bond.

In some embodiments, the surface of each pillar is square or rectangularin shape. In some embodiments, the substrate can be coupled to a silicondioxide layer. The silicon dioxide layer can be about 0.5 μm to 3 μmthick. In some embodiments, the substrate can be coupled to a wafer,e.g., a silicon wafer. The silicon dioxide layer can be about 700 μm to750 μm thick.

Arrays

Also disclosed herein are arrays. In some embodiments, the surface ofthe array is functionalized with free carboxylic acids. In someembodiments, the free carboxylic acids are activated to bind to aminegroups, e.g., during polypeptide synthesis on the surface of the array.In some embodiments, the surface density of free carboxylic acid groupson the array is greater than 10/cm², 100/cm², 1,000/cm², 10,000/cm²,100,000/cm², 1,000,000/cm², or 10,000,000/cm².

In some embodiments, an array can be a three-dimensional array, e.g., aporous array comprising features attached to the surface of the porousarray. In some embodiments, the surface of a porous array includesexternal surfaces and surfaces defining pore volume within the porousarray. In some embodiments, a three-dimensional array can includefeatures attached to a surface at positionally-defined locations, saidfeatures each comprising: a collection of peptide chains of determinablesequence and intended length. In one embodiment, within an individualfeature, the fraction of peptide chains within said collection havingthe intended length is characterized by an average coupling efficiencyfor each coupling step of greater than 98%.

In some embodiments, the average coupling efficiency for each couplingstep is at least 98.5%. In some embodiments, the average couplingefficiency for each coupling step is at least 99%. In some embodiments,the average coupling efficiency for each coupling step is at least 90,91, 92, 93, 94, 95, 96, 97, 98, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0,99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100%.

In some embodiments, each peptide chain is from 5 to 60 amino acids inlength. In some embodiments, each peptide chain is at least 5 aminoacids in length. In some embodiments, each peptide chain is at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. Insome embodiments, each peptide chain is less than 5, at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, orgreater than 60 amino acids in length. In some embodiments, each peptidechain comprises one or more L amino acids. In some embodiments, eachpeptide chain comprises one or more D amino acids. In some embodiments,each peptide chain comprises one or more naturally occurring aminoacids. In some embodiments, each peptide chain comprises one or moresynthetic amino acids.

In some embodiments, an array can include at least 1,000 differentpeptide chains attached to the surface. In some embodiments, an arraycan include at least 10,000 different peptide chains attached to thesurface. In some embodiments, an array can include at least 100, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or greaterthan 10,000 different peptide chains attached to the surface (or anyinteger in between).

In some embodiments, each of the positionally-defined locations is at adifferent, known location that is physically separated from each of theother positionally-defined locations. In some embodiments, each of thepositionally-defined locations is a positionally-distinguishablelocation. In some embodiments, each determinable sequence is a knownsequence. In some embodiments, each determinable sequence is a distinctsequence.

In some embodiments, the features are covalently attached to thesurface. In some embodiments, said peptide chains are attached to thesurface through a linker molecule or a coupling molecule.

In some embodiments, the features comprise a plurality of distinct,nested, overlapping peptide chains comprising subsequences derived froma source protein having a known sequence. In some embodiments, eachpeptide chain in the plurality is substantially the same length. In someembodiments, each peptide chain in the plurality is the same length. Insome embodiments, each peptide chain in the plurality is at least 5amino acids in length. In some embodiments, each peptide chain in theplurality is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60amino acids in length. In some embodiments, each peptide chain in theplurality is less than 5, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greater than 60 amino acidsin length. In some embodiments, at least one peptide chain in theplurality is at least 5 amino acids in length. In some embodiments, atleast one peptide chain in the plurality is at least 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 amino acids in length. In someembodiments, at least one peptide chain in the plurality is less than 5,at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, or greater than 60 amino acids in length. In someembodiments, each polypeptide in a feature is substantially the samelength. In some embodiments, each polypeptide in a feature is the samelength. In some embodiments, the features comprise a plurality ofpeptide chains each having a random, determinable sequence of aminoacids.

Methods

Methods of Manufacturing Substrates

Also disclosed herein are methods for making substrates. In someembodiments, a method of producing a substrate can include coupling aporous layer to a support layer. The support layer can comprise anymetal or plastic or silicon or silicon oxide or silicon nitride. In oneembodiment, the substrate comprises multiple carboxylic acid substratesattached to the substrate for binding peptides during peptide synthesisand protein coupling. In some embodiments, a method of producing asubstrate can include coupling a porous layer to a plurality of pillars,wherein the porous layer comprises functional groups for attachment of acompound to the substrate, wherein the plurality of pillars are coupledto a planar layer in positionally-defined locations, wherein each pillarhas a planar surface extended from the planar layer, wherein thedistance between the surface of each pillar and the upper surface of theplanar layer is between about 1,000-5,000 angstroms, and wherein theplurality of pillars are present at a density of greater than about10,000/cm².

In some embodiments, the surface of each pillar is parallel to the uppersurface of the planar layer. In some embodiments, the surface of eachpillar is substantially parallel to the upper surface of the planarlayer.

In some embodiments, a method of preparing a substrate surface caninclude obtaining a surface comprising silicon dioxide and contactingthe surface with a photoactive coupling formulation comprising aphotoactive compound, a coupling molecule, a coupling reagent, apolymer, and a solvent; and applying ultraviolet light topositionally-defined locations located on the top of the surface and incontact with the photoactive formulation.

Methods of Manufacturing Arrays

Also disclosed herein are methods for manufacturing arrays. In someembodiments, the arrays disclosed herein can be synthesized in situ on asurface, e.g., a substrate disclosed herein. In some instances, thearrays are made using photolithography. For example, the substrate iscontacted with a photoactive coupling solution. Masks can be used tocontrol radiation or light exposure to specific locations on a surfaceprovided with free linker molecules or free coupling molecules havingprotecting groups. In the exposed locations, the protecting groups areremoved, resulting in one or more newly exposed reactive moieties on thecoupling molecule or linker molecule. The desired linker or couplingmolecule is then coupled to the unprotected attached molecules, e.g., atthe carboxylic acid group. The process can be repeated to synthesize alarge number of features in specific or positionally-defined locationson a surface (see, for example, U.S. Pat. No. 5,143,854 to Pirrung etal., U.S. Patent Application Publication Nos. 2007/0154946 (filed onDec. 29, 2005), 2007/0122841 (filed on Nov. 30, 2005), 2007/0122842(filed on Mar. 30, 2006), 2008/0108149 (filed on Oct. 23, 2006), and2010/0093554 (filed on Jun. 2, 2008), each of which is hereinincorporated by reference).

In some embodiments, a method of producing a three-dimensional (e.g.,porous) array of features, can include obtaining a porous layer attachedto a surface; and attaching the features to the porous layer, saidfeatures each comprising a collection of peptide chains of determinablesequence and intended length, wherein within an individual feature, thefraction of peptide chains within said collection having the intendedlength is characterized by an average coupling efficiency for eachcoupling step of at least about 98%. In some embodiments, the featuresare attached to the surface using a photoactive coupling formulation,comprising a photoactive compound, a coupling molecule, a couplingreagent, a polymer, and a solvent. In some embodiments, the features areattached to the surface using a photoactive coupling formulationdisclosed herein. In some embodiments, the photoactive couplingformulation is stripped away using water.

In one embodiment, described herein is a process of manufacturing anarray. A surface comprising attached carboxylic acid groups is provided.The surface is contacted with a photoactive coupling solution comprisinga photoactive compound, a coupling molecule, a coupling reagent, apolymer, and a solvent. The surface is exposed to ultraviolet light in adeep ultra violet scanner tool according to a pattern defined by aphotomask, wherein the locations exposed to ultraviolet light undergophoto base generation due to the presence of a photobase generator inthe photoactive coupling solution. The expose energy can be from 1mJ/cm² to 100 mJ/cm² in order to produce enough photobase.

The surface is post baked upon exposure in a post exposure bake module.Post exposure bake acts as a chemical amplification step. The bakingstep amplifies the initially generated photobase and also enhances therate of diffusion to the substrate. The post bake temperature can varybetween 75° C. to 115° C., depending on the thickness of the poroussurface, for at least 60 seconds and not usually exceeding 120 seconds.The free carboxylic acid group is coupled to the deprotected amine groupof a free peptide or polypeptide, resulting in coupling of the freepeptide or polypeptide to the carboxylic acid group attached to thesurface. This surface may be a porous surface. The synthesis of peptidescoupled to a carboxylic acid group attached to the surface occurs in anN→C synthesis orientation, with the amine group of free peptidesattaching to carboxylic acid groups bound to the surface of thesubstrate. Alternatively, a diamine linker may be attached to a freecarboxylic acid group to orient synthesis in a C→N direction, with thecarboxylic acid group of free peptides attaching to amine groups boundto the surface of the substrate.

The photoactive coupling solution can now be stripped away. In someembodiments, provided herein is a method of stripping the photoresistcompletely with deionized (DI) water. This process is accomplished in adeveloper module. The wafer is spun on a vacuum chuck for, e.g., 60seconds to 90 seconds and deionized water is dispensed through a nozzlefor about 30 seconds.

The photoactive coupling formulation can be applied to the surface in acoupling spin module. A coupling spin module can typically have 20nozzles or more to feed the photoactive coupling formulation. Thesenozzles can be made to dispense the photoactive coupling formulation bymeans of pressurizing the cylinders that hold these solutions or by apump that dispenses the required amount. In some embodiments, the pumpis employed to dispense 5-8 cc of the photoactive coupling formulationonto the substrate. The substrate is spun on a vacuum chuck for 15-30seconds and the photoactive coupling formulation is dispensed. The spinspeed can be set to 2000 to 2500 rpm.

Optionally, a cap film solution coat is applied on the surface toprevent the unreacted amino groups on the substrate from reacting withthe next coupling molecule. The cap film coat solution can be preparedas follows: a solvent, a polymer, and a coupling molecule. The solventthat can be used can be an organic solvent like N methyl pyrrolidone,dimethyl formamide, or combinations thereof. The capping molecule istypically acetic anhydride and the polymer can be polyvinyl pyrrolidone,polyvinyl alcohol, polymethyl methacrylate, poly (methyl iso propenyl)ketone, or poly (2 methyl pentene 1 sulfone). In some embodiments, thecapping molecule is ethanolamine.

This process is done in a capping spin module. A capping spin module caninclude one nozzle that can be made to dispense the cap film coatsolution onto the substrate. This solution can be dispensed throughpressurizing the cylinder that stores the cap film coat solution orthrough a pump that precisely dispenses the required amount. In someembodiments, a pump is used to dispense around 5-8 cc of the cap coatsolution onto the substrate. The substrate is spun on a vacuum chuck for15-30 s and the coupling formulation is dispensed. The spin speed can beset to 2000 to 2500 rpm.

The substrates with the capping solution are baked in a cap bake module.A capping bake module is a hot plate set up specifically to receivewafers just after the capping film coat is applied. In some embodiments,provided herein is a method of baking the spin coated capping coatsolution in a hot plate to accelerate the capping reactionsignificantly. Hot plate baking generally reduces the capping time foramino acids to less than two minutes.

The byproducts of the capping reaction are stripped in a strippermodule. A stripper module can include several nozzles, typically up to10, set up to dispense organic solvents such as acetone, iso propylalcohol, N methyl pyrrolidone, dimethyl formamide, DI water, etc. Insome embodiments, the nozzles can be designated for acetone followed byiso propyl alcohol to be dispensed onto the spinning wafer. The spinspeed is set to be 2000 to 2500 rpm for around 20 s.

This entire cycle can be repeated as desired with different couplingmolecules each time to obtain a desired sequence.

In some embodiments, an array comprising a surface of free carboxylicacids is used to synthesize polypeptides in an N→C orientation. In oneembodiment, the carboxylic acids on the surface of the substrate areactivated (e.g., converted to a carbonyl) to allow them to bind to freeamine groups on an amino acid. In one embodiment, activation ofcarboxylic acids on the group of the surface can be done by addition ofa solution comprising a carbodiimide or succinimide to the surface ofthe array. In some embodiments, carboxylic acids can be activated byaddition of a solution comprising1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC],N-hydroxysuccinimide [NHS], 1,3-diisopropylcarbodiimide [DIC],hydroxybenzotriazole (HOBt),(0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) [HATU],benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP], or N,N-diisopropylethylamine [DIEA] to the surface of thearray. The activation solution is washed away and the surface of thearray is prepared for addition of an amino acid layer (i.e., one aminoacid at each activated carboxylic acid group). Carboxylic acid groupsremain activated for up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

Addition of a solution comprising an amino acid with a free amine groupto the activated carboxylic acid surface of the array results in bindingof a single amino acid to each carboxylic acid group. In someembodiments, the amino acid comprises an amino acid with protected aminegroups. Using a photosensitive chemical reaction, the protecting groupcan be removed from the amine group of selected amino acids atsite-specific locations using a reticle. For example, Fmoc-protectedamino acids are mixed in a solution comprising a photobase generator.Upon exposure of the solution on the array to a specific frequency oflight at site-specific locations, the photobase generator will release abase which will deprotect the amino acid, resulting in coupling of theamino acid to the activated carboxylic acid group on the surface of thearray. Another method involves using a protected base that is thenunprotected by a photoacid released by a photoacid generator upon lightexposure. In some embodiments, the protected base is N-Boc-piperidine or1,4-bis(N-Boc)-piperazine.

After a completed layer of amino acids is coupled, remaining uncoupledactivated carboxylic acids are capped to prevent nonspecific binding ofamino acids on subsequent synthesis steps. The steps of activation,addition of an amino acid layer, and capping are repeated as necessaryto synthesize the desired polypeptides at specific locations on thearray.

In one embodiment, peptides synthesized in the N→C terminus directioncan be capped with a diamine molecule to enhance binding properties ofselected polypeptide sequences to a biological molecule, e.g., anantibody. In other embodiments, peptides synthesized in the C→Ndirection can be capped with a dicarboxylic acid molecule to enhancebinding properties of selected sequences to a biological molecule.

While synthesizing polypeptides in parallel on the surface of an array,the method described herein ensures complete activation of carboxylicacid on the surface of the array. Due to stability of the activatedester for an extended period of time, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more couplingcycles may be completed after a single activation step (e.g., to couplean entire layer of 2-25 or more different amino acids at differentlocations on the array). As the coupling occurs during hard bake(heating in a hot plate at 85-90° Celsius for 90 seconds immediatelyafter coating) and due to the presence of excess amino acid in thesolution, complete 100% deprotection of Fmoc-protected amino acid maynot be required for significantly high coupling yields. After additionof all amino acids and capping, all free activated carboxylic acids areeither coupled or capped, thus resulting in high efficiency and accuracyof polypeptide synthesis.

Methods of Use of Peptide Arrays

Also disclosed herein are methods of using substrates, formulations,and/or arrays. Uses of the arrays disclosed herein can include researchapplications, therapeutic purposes, medical diagnostics, and/orstratifying one or more patients.

Any of the arrays described herein can be used as a research tool or ina research application. In one aspect, arrays can be used for highthroughput screening assays. For example, enzyme substrates (i.e.,peptides on a peptide array described herein) can be tested bysubjecting the array to an enzyme and identifying the presence orabsence of enzyme substrate(s) on the array, e.g., by detecting at leastone change among the features of the array.

Arrays can also be used in screening assays for ligand binding, todetermine substrate specificity, or for the identification of peptidesthat inhibit or activate proteins. Labeling techniques, protease assays,as well as binding assays useful for carrying out these methodologiesare generally well-known to one of skill in the art.

In some embodiments, an array can be used to represent a known proteinsequence as a sequence of overlapping peptides. For example, the aminoacid sequence of a known protein is divided into overlapping sequencesegments of any length and of any suitable overlapping frame, andpeptides corresponding to the respective sequence segments are in-situsynthesized as disclosed herein. The individual peptide segments sosynthesized can be arranged starting from the amino terminus of theknown protein.

In some embodiments, an array is used in a method wherein the antigenicrepresentation of the array includes at least one region where the wholeantigen sequence of a known protein is spanned via epitope sliding; theimmunoactive regions of the antigen are determined by contacting one ormore clinical samples on the array or a plurality of different arrays,and the set of peptide sequences required to represent the known proteinantigen are reduced.

In some embodiments, a sample is applied to an array having a pluralityof random peptides. The random peptides can be screened and BLASTed todetermine homologous domains with, e.g., a 90% or more identity to agiven antigenic sequence. In some aspect, the whole antigenic sequencecan then be synthesized and used to identify potential markers and/orcauses of a disease of interest.

In some embodiments, an array is used for high throughput screening ofone or more genetic factors. Proteins associated with a gene can be apotential antigen and antibodies against these proteins can be used toestimate the relation between gene and a disease.

In another example, an array can be used to identify one or morebiomarkers. Biomarkers can be used for the diagnosis, prognosis,treatment, and management of diseases. Biomarkers may be expressed, orabsent, or at a different level in an individual, depending on thedisease condition, stage of the disease, and response to diseasetreatment. Biomarkers can be, e.g., DNA, RNA, proteins (e.g., enzymessuch as kinases), sugars, salts, fats, lipids, or ions.

Arrays can also be used for therapeutic purposes, e.g., identifying oneor more bioactive agents. A method for identifying a bioactive agent cancomprise applying a plurality of test compounds to an array andidentifying at least one test compound as a bioactive agent. The testcompounds can be small molecules, aptamers, oligonucleotides, chemicals,natural extracts, peptides, proteins, fragment of antibodies, antibodylike molecules or antibodies. The bioactive agent can be a therapeuticagent or modifier of therapeutic targets. Therapeutic targets caninclude phosphatases, proteases, ligases, signal transduction molecules,transcription factors, protein transporters, protein sorters, cellsurface receptors, secreted factors, and cytoskeleton proteins.

In another aspect, an array can be used to identify drug candidates fortherapeutic use. For example, when one or more epitopes for specificantibodies are determined by an assay (e.g., a binding assay such as anELISA), the epitopes can be used to develop a drug (e.g., a monoclonalneutralizing antibody) to target antibodies in disease.

In one aspect, also provided are arrays for use in medical diagnostics.An array can be used to determine a response to administration of drugsor vaccines. For example, an individual's response to a vaccine can bedetermined by detecting the antibody level of the individual by using anarray with peptides representing epitopes recognized by the antibodiesproduced by the induced immune response. Another diagnostic use is totest an individual for the presence of biomarkers, wherein samples aretaken from a subject and the sample is tested for the presence of one ormore biomarkers.

Arrays can also be used to stratify patient populations based upon thepresence or absence of a biomarker that indicates the likelihood asubject will respond to a therapeutic treatment. The arrays can be usedto identify known biomarkers to determine the appropriate treatmentgroup. For example, a sample from a subject with a condition can beapplied to an array. Binding to the array may indicate the presence of abiomarker for a condition. Previous studies may indicate that thebiomarker is associated with a positive outcome following a treatment,whereas absence of the biomarker is associated with a negative orneutral outcome following a treatment. Because the patient has thebiomarker, a health care professional may stratify the patient into agroup that receives the treatment.

In some embodiments, a method of detecting the presence or absence of aprotein of interest (e.g., an antibody) in a sample can includeobtaining an array disclosed herein and contacted with a samplesuspected of comprising the protein of interest; and determining whetherthe protein of interest is present in the sample by detecting thepresence or absence of binding to one or more features of the array. Insome embodiments, the protein of interest can be obtained from a bodilyfluid, such as amniotic fluid, aqueous humour, vitreous humour, bile,blood serum, breast milk, cerebrospinal fluid, cerumen, chyle,endolymph, perilymph, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus, peritoneal fluid, pleural fluid, pus, saliva,sebum, semen, sweat, synovial fluid, tears, vaginal secretion, vomit, orurine.

In some embodiments, a method of identifying a vaccine candidate caninclude obtaining an array disclosed herein contacted with a samplederived from a subject previously administered the vaccine candidate,wherein the sample comprises a plurality of antibodies; and determiningthe binding specificity of the plurality of antibodies to one or morefeatures of the array. In some embodiments, the features comprise aplurality of distinct, nested, overlapping peptide chains comprisingsubsequences derived from a source protein having a known sequence.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Compound Examples Example 11-(diethylamino-methyl)-4-phenyl-1,4-dihydro-5H-tetrazole-5-thione

1-(diethylamino-methyl)-4-phenyl-1,4-dihydro-5H-tetrazole-5-thione iscommercially available from Sigma Aldrich.

Example 21-(3-(diethylamino)-propyl)-4-(2-methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione

1-(3-(diethylamino)-propyl)-4-(2-methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thionewas prepared according to Scheme 1.

¹H NMR (400 MHz, CDCl3): 7.47-7.38 (m, 2H), 7.01-6.95 (m, 2H), 4.43 (t,2H), 3.83 (s, 3H), 2.62-2.54 (m, 6H), 2.14-2.11 (m, 2H), 1.07-1.04 (t,6H). MS, m/z, calculated for C₁₅H₂₃N₅OS [MH⁺] 322.44, observed 322.

Example 3 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane

1,3-Bis[(2-nitrobenzyl)oxy carbonyl-4-piperidyl]propane is commerciallyavailable from Sigma Aldrich.

Example 4 1,3-Bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane

1,3-Bis[1-(9-fluorenylmethoxy carbonyl)-4-piperidyl]propane iscommercially available from Sigma Aldrich.

Example 5 1-Phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane) Bromide

To a solution of the 2-bromoacetophenone in toluene one equivalent ofethereal solution of 1,4-diazabicyclo[2.2.2]octane was added at roomtemperature. The reaction mixture was stirred at room temperature forone hour. The precipitated bromide was filtered, washed with diethylether thrice and dried to give the title compound in 91% yield.

¹H NMR (400 MHz, D₂O): 7.88 (d, 2H, ArH), 7.67 (t, 1H, ArH), 7.50 (t,2H, ArH), 4.70 (s, 2H, CH₂), 3.70 (t, 6H, NCH₂), 3.20 (t, 6H, NCH₂).

Example 61-Phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane)tetraphenylborate

To an aqueous solution of the1-phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane) bromide one equivalentof an aqueous solution of sodium tetraphenylborate was added. Thereaction mixture was stirred for one hour. The solid was filtered,washed with water, ether and dried to give the title compound in 40%yield.

¹H NMR (400 MHz, DMSO-d₆): 8.00 (d, 2H, ArH), 7.75 (t, 1H, ArH), 7.62(t, 2H, ArH), 7.18-7.16 (br s, 8H, ArH), 6.92 (t, 8H, ArH), 6.79 (t, 4H,ArH), 4.70 (s, 2H, CH₂), 3.58 (t, 6H, NCH₂), 3.12 (t, 6H, NCH₂).

Example 7 1-Naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane) Bromide

To a solution of the 2-bromo-2′-acetonaphthone in toluene one equivalentof ethereal solution of 1,4-diazabicyclo[2.2.2]octane was added at roomtemperature. The reaction mixture was stirred at room temperature forone hour. The precipitated bromide was filtered, washed with diethylether till the filtrate was colorless and dried to give the titlecompound in 91% yield.

¹H NMR (400 MHz, D₂O): 8.44 (br s, 1H, ArH), 7.99-7.89 (m, 3H, ArH),7.83 (dd, 1H, ArH), 7.67-7.62 (m, 1H, ArH), 7.60-7.56 (m, 1H, ArH), 4.70(s, 2H, CH₂), 3.72 (t, 6H, NCH₂), 3.21 (t, 6H, NCH₂).

Example 8 1-Naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane)Tetraphenylborate

To an aqueous solution of the1-naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane) bromide oneequivalent of an aqueous solution of sodium tetraphenylborate was added.The reaction mixture was stirred for one hour. The solid was filtered,washed with water, ether and dried to give the title compound in 88%yield.

¹H NMR (400 MHz, DMSO-d₆): 8.74 (br s, 1H, ArH), 8.17-8.11 (m, 2H, ArH),8.07-8.00 (m, 2H, ArH), 7.77-7.70 (m, 2H, ArH), 7.25-7.23 (m, 1H, ArH),7.18-7.15 (m, 8H, ArH), 6.92 (t, 8H, ArH), 6.79 (t, 4H, ArH), 5.31 (s,2H, CH₂), 3.63 (t, 6H, NCH₂), 3.16 (t, 6H, NCH₂).

Example 9 1,5,7-triazabicyclo[4.4.0]dec-5-enylphenylglyoxylate

A solution of the phenylglyoxylic acid (0.25 g, 1.66 mmol) and1,5,7-triazabicyclo[4.4.0]dec-5-ene (0.24 g 1.75 mmol) were stirred inethanol at room temperature for 18 hours. Evaporation of solvent undervacuum yielded a solid that was recrystallized from hexane/ethanol togive the title compound (0.32 g) in 66% yield.

¹H NMR (400 MHz, DMSO-d₆): 8.63 (s, 2H), 7.83 (d, 2H), 7.61-7.57 (m,1H), 7.51-7.48 (m, 2H), 3.28 (t, 4H), 3.18 (t, 4H), 1.91-1.85 (m, 4H).

Example 10 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-4-nitrophenylglyoxylate

A solution of the 4-nitrophenylglyoxylic acid (0.25 g, 1.28 mmol) and1,5,7-triazabicyclo[4.4.0]dec-5-ene (0.0.18 g 1.34 mmol) were stirred inethanol at room temperature. A solid precipitated out, washed withhexane and recrystallized from hexane/ethanol to give the title compound(0.3 g) in 70% yield.

¹H NMR (400 MHz, DMSO-d₆): 8.34-8.32 (m, 2H), 8.07-8.04 (m, 2H), 7.99(br s, 2H), 3.28 (t, 4H), 3.18 (t, 4H), 1.91-1.85 (m, 4H).

Example 11 1,5,7-triazabicyclo[4.4.0]dec-5-enyltetraphenylborate

1,5,7-Triazabicyclo[4.4.0]dec-5-ene (61 mmol) was dissolved in 61 mL of10% HCl (aq), followed by addition of a suspension of NaBPh₄ (67 mmol,1.1 equivalent) in 85 ml water. A white precipitate was formed that wasfiltered and washed several times with water, methanol, and diethylether. The solid obtained was dried under vacuum to give the titlecompound in 82% yield.

¹H NMR (400 MHz, DMSO-d₆): 7.40 (s, 2H), 7.20-7.17 (m, 8H), 6.93 (t,8H), 6.80 (t, 4H), 3.25 (t, 4H), 3.17 (t, 4H), 1.88-1.83 (m, 4H).

Example 12 1,8-Diazabicyclo[5.4.0]undec-7-enyltetraphenylborate

1,8-Diazabicyclo[5.4.0]undec-7-ene (9.85 mmol) was dissolved in 10 mL of10% HCl (aq), followed by addition of a suspension of NaBPh₄ (10.85mmol, 1.1 equivalent) in 13 ml water. A white precipitate was formedthat was filtered and washed several times with water, methanol anddiethyl ether. The solid obtained was dried under vacuum to give thetitle compound in 64% yield.

¹H NMR (400 MHz, DMSO-d₆): 9.48 (s, 1H), 7.20-7.16 (m, 8H), 6.93 (t,8H), 6.80 (t, 4H), 3.55-3.52 (m, 2H), 3.46 (t, 2H), 3.24 (t, 2H),2.64-2.61 (m, 2H), 1.93-1.87 (m, 2H) 1.67-1.60 (m, 6H).

Array Examples Example 13: Production of a COOH Coated Substrate UsingBis-Polyethylene Glycol Carboxy Methyl Ether

This example describes how to construct a substrate comprising COOHgroups. Silicon wafers deposited with Nickel 1000 Å on a siliconsubstrate were obtained from University Wafers. Dextran Bio Xtra(MW40000) was obtained from Sigma Aldrich. Bis-Polyethylene glycolcarboxy methyl ether was obtained from Sigma Aldrich. Poly vinylpyrollidone 1000000 was obtained from Poly Sciences Inc. The above threepolymers were dissolved in a solvent composition of 50% Ethyllactate/50% water by weight in a ratio of 2:2:1 by weight along with 2%by weight photoacid generator dimethyl-2,4-dihydroxyphenylsulfoniumtriflate obtained from Oakwood Chemicals Inc. This solution wasspin-coated onto the silicon wafer.

The coated silicon wafer was spun at 3000 rpm to obtain a uniform coatof thickness 100 nm. The wafer was then exposed in a deep UV scannerNikon S 203 at 250 mJ/cm² and then baked at 65° Celsius for 90 secondsin a hot plate. The coating was then stripped off the wafer with acetoneand isopropyl alcohol followed by a deionized water rinse. The substratehad a matrix of free COOH groups ready to be activated and coupled witha protein or an amino acid for peptide synthesis. The 2-dimensionalconcentration of COOH groups along the layer is increased on a porousdextran substrate as compared to a planar substrate.

Example 14: Production of a COOH Coated Substrate Using Silane-PEG-COOH

Production of a COOH coated substrate was performed as follows:Silane-PEG-COOH was obtained from Nanocs. Pure ethyl alcohol wasobtained from EMD Millipore. A mixture of 99.5% by weight ethyl alcoholand 0.5% by weight of Silane-PEG-COOH was dissolved and layered on asilica wafer for 48 hours at room temperature. The silica wafer was thenwashed with ethyl alcohol for 5 minutes followed by washing withdeionized water for 5 minutes.

Example 15: Production of a COOH Coated Substrate Using SuccinicAnhydride

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110 Celsius nitrogenbake oven to grow a mono silane layer with a —NH₂ group to attach alinker molecule.

Production of a COOH coated substrate was performed as follows: SuccinicAnhydride was obtained from Sigma-Aldrich. N,N-dimethylformamide [DMF]was obtained from VWR International. A mixture of 50% by weight DMF and50% by weight Succinic Anhydride was dissolved and reacted with a silicawafer containing NH₂ surface for 48 hours. The wafer was then washedwith DMF for 5 minutes followed by washing with deionized water for 5minutes.

Example 16: Production of a COOH Coated Substrate Using PEG Diacid

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110 Celsius nitrogenbake oven to grow a mono silane layer with a —NH₂ group to attach alinker molecule.

Production of a COOH coated substrate was performed as follows:Poly(ethylene glycol) diacid (i.e., PEG-dipropionic acid) was obtainedfrom Sigma-Aldrich. PEG diacid comprises 2 carboxylic acid groups.1,3-diisopropylcarbodiimide [DIC] was obtained from Advanced ChemTech.Hydroxybenzotriazole [HOBt] was obtained from Anaspec.N-Methyl-2-Pyrrolidone [NMP] was obtained from VWR International. Amixture comprising of 2% by weight of DIC, 1% by weight of HOBt, 1% byweight of Poly(ethylene glycol) diacid dissolved in NMP was reacted withthe silica wafer containing an NH₂ surface for 60 minutes. The wafer wasthen washed with NMP for 5 minutes. This was followed by reaction with acapping solution containing 50% Acetic Anhydride and 50% NMP to reactwith the unreacted NH₂ remaining on the surface for 15 minutes. This wasfollowed by washing the wafer in NMP for 5 minutes.

Example 17: Production of a COOH Coated Substrate Using Trimesic Acid

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110° Celsius nitrogenbake oven to grow a mono silane layer with a —NH₂ group to attach alinker molecule.

Production of a COOH coated substrate was performed as follows: Trimesicacid (Benzene-1,3,5-tricarboxylic acid, H3BTC) [TMA] was obtained fromSigma-Aldrich. Trimesic acid comprises 3 carboxylic acid groups. Amixture comprising of 2% by weight of DIC, 1% by weight of HOBt, 1% byweight of TMA dissolved in NMP was reacted with the silica wafercontaining NH₂ surface for 12 hours. The wafer was then washed with NMPfor 5 minutes. This was followed by reaction with a capping solutioncontaining 50% by weight Acetic Anhydride and 50% by weight NMP to reactwith the unreacted NH₂ remaining on the surface for 15 minutes. This wasfollowed by washing the wafer in NMP for 5 minutes.

Example 18: Production of a COOH Coated Substrate Using Mellitic Acid

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110° C. nitrogen bakeoven to grow a mono silane layer with a —NH₂ group to attach a linkermolecule.

Production of a COOH coated substrate was performed as follows: Melliticacid (Benzenehexacarboxylic acid) [MA] was obtained from Sigma Aldrich.Mellitic acid comprises 6 carboxylic acid groups. A mixture comprisingof 2% by weight of DIC, 1% by weight of HOBt, 1% by weight of MAdissolved in NMP was reacted with the silica wafer containing NH₂surface for 8 hours. The wafer was then washed with NMP for 5 minutes.This was followed by reaction with a capping solution containing 50% byweight Acetic Anhydride and 50% by weight NMP to react with theunreacted NH₂ remaining on the surface for 15 minutes. This was followedby washing the wafer in NMP for 5 minutes.

Example 19: Production of a COOH Coated Substrate Using Dextran andBenzophenone (Dextran 1)

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110° Celsius nitrogenbake oven to grow a mono silane layer with a —NH₂ group to attach alinker molecule.

Production of a 3-dimensional COOH coated substrate was performed asfollows: CM-Dextran (i.e., carboxy methyl dextran) salt was obtainedfrom Sigma Aldrich. Benzophenone-4-carboxylic acid and4-Aminobenzophenone were obtained from Sigma Aldrich. A mixture of 4% byweight EDC, 2% by weight of NHS and 2.5% by weight ofbenzophenone-4-carboxylic acid dissolved in ethanol was reacted with asilica wafer containing NH₂ surface for 60 minutes. A solutioncontaining 3% by weight 4-Aminobenzophenone and 2% by weight of CMDextran was generated by mixing with each other in solution phase for120 minutes in the presence of EDC and NHS. EDC and NHS activated theCOOH on the CM dextran, allowing coupling of the activated carboxylicacid group to the 4-aminobenzophenone. The resulting solution was thenfiltered to select for the portion containing coupled aminobenzophenoneand CM Dextran. A solution comprising the portion containing coupledaminobenzophenone and CM Dextran along with a suitable polymer was thenspin-coated onto the wafer reacted with benzophenone previously andexposed at 248 nm. Benzophenone on the wafer surface coupled with thebenzophenone in solution which was coupled to CM Dextran. This linkedthe CM Dextran to the array surface via a benzophenone-benzophenoneinteraction, thus creating a substrate with a 3-dimensional arrangementof carboxylic acids on the surface.

Example 20: Production of a COOH Coated Substrate Using Dextran and anAmine Surface (Dextran 2)

Wafer with an NH₂ surface was prepared as follows: Aminopropyl triethoxysilane (APTES) was obtained from Sigma Aldrich. 100% Ethanol wasobtained from VWR. The wafers were first washed with ethanol for 5minutes and then in 1% by weight APTES/Ethanol for 20-30 minutes to growthe silane layer. Then the wafers were cured in a 110° Celsius nitrogenbake oven to grow a mono silane layer with a —NH₂ group to attach alinker molecule.

Production of a 3-dimensional COOH coated substrate was performed asfollows: A mixture comprising of 2% by weight of DIC, 1% by weight ofHOBt, 2.5% by weight of CM Dextran (i.e., carboxy methyl dextran)dissolved in NMP was reacted with the silica wafer containing NH₂surface for 60 minutes. The wafer was then washed with NMP for 5minutes. This was followed by reaction with a capping solutioncontaining 50% by weight acetic anhydride and 50% by weight NMP to capthe unreacted NH₂ remaining on the surface for 15 minutes. This wasfollowed by washing the wafer in NMP for 5 minutes. This created asubstrate with a 3-dimensional arrangement of carboxylic acids on thesurface.

Example 21: Carboxyl Surface Density on COOH Coated Substrates

Wafers with carboxyl surface were derivatized using different methodsdescribed in Examples 14-20 (Example 14: Silane PEG COOH, Example 15:Succinic Anhydride, Example 16: PEG diacid, Example 17: Trimesic acid,Example 18: Mellitic acid, Example 19: Dextran 1, and Example 20:Dextran 2). Surface density of the array generated by each method wastested. 4′-(Aminomethyl) Fluorescein, Hydrochloride was obtained fromLife Technologies. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC]and N-hydroxysuccinimide [NHS] were obtained from Sigma Aldrich. Thecarboxyl surface of each array was activated with an activation mixtureof 4% by weight EDC and 2% by weight NHS dissolved in deionized waterfor 10 minutes. This was followed by washing the carboxyl surface ofeach array with deionized water for 3 minutes. A mixture containing 1%by weight of 4′-(Aminomethyl) fluorescein dissolved in deionized waterwas then added to the array and allowed to react for 30 minutes. Thiswas followed by washing the array with deionized water for 5 minutes.Intensity of fluorescein was then checked using a 488 nm laser for allCOOH substrates. The resulting fluorescein intensity correlating tocarboxyl surface density is shown in FIG. 1.

Peptide synthesis and antibody binding as described in the methods belowwere performed. Results indicated a higher density of peptidessynthesized on the 3-dimensional COOH surfaces generated in Example 19and 20 (i.e., Dextran 1 and Dextran 2; data not shown).

Example 22: Production of a Substrate with Pillars

This example describes how to construct a substrate comprising pillars.Silicon wafers with 2.4 μm thermally grown oxide were obtained fromUniversity Wafers. The surface of the silicon wafer was cleaned withdeionized water to remove contaminants from the wafer surface. Thesurface of the silicon wafer was primed for chemical adhesion of anorganic compound to the wafer by applying vapors of hexamethyldisilizane(HMDS) onto a heated wafer substrate using a spray module at 200-220°Celsius for 30-50 seconds. HMDS was obtained from Sigma Aldrich Inc.HMDS acts as a “bridge” with properties to bind to both the wafersurface and the photoresist. The wafers were spun coat in a photoresistcoat module with a commercially available deep Ultra violet photoresist,P5107 obtained from Rohm and Haas or AZ DX7260p 700 from AZ ElectronicMaterials, to obtain a thickness of 6000 Å. The wafers were then bakedin a hot plate at 120° Celsius for 60 seconds.

Photomasks that have the patterned regions to create the features wereused to image the array on to the substrate surface. The wafers werethen exposed in a 248 nm deep ultra violet radiation scanner tool, Nikon5203, with expose energy of 18 mJ/cm². The wafers were thenpost-exposure baked at 1100 Celsius for 120 seconds in a hot plate anddeveloped with commercially available NMD-3 developer, obtained fromTokyo Ohka Kogyo Co., Ltd., for 60 seconds.

After this the oxide was etched by using either a wet etch process ordry plasma etch process. Standard semiconductor etch techniques wereused. Oxide etch depths were from 1000 Å to 2000 Å.

After etching, chromium was deposited to a thickness of 500 Å to 1500 Åby a physical deposition method. Standard etching and metal depositiontechniques were employed.

After the chromium was deposited, the resist was lifted off with thefollowing process: The wafers were left in Nanostrip obtained fromCyantek Inc. overnight and then dipped in Piranha solution for 90 min.Piranha solution is a 50:50 mixture of sulfuric acid and hydrogenperoxide. Sulfuric acid and hydrogen peroxide were obtained from SigmaAldrich Corp. Plasma ashing was performed to oxidize the remainingimpurities. This process produced a substrate having pillars of silicondioxide separated by metal.

Alternatively, the deposited chromium was also polished to a depth of500 Å to 1500 Å, depending on the deposition. The polishing wasperformed to obtain pillars of silicon dioxide separated by metal.

Derivatization: The wafers were then surface derivatized using themethods provided in Examples 13-21 to coat the pillar surface with freecarboxylic acid attachment groups (i.e., COOH groups).

Example 23: Synthesis of Homopolymers and Heteropolymers fromFmoc-Protected Amino Acids

This example illustrates the method of C→N synthesis of peptides on achip array using carbodiimide activation of free carboxylic acid groups.Wafers with COOH groups were prepared as explained in Example 13. COOHgroups were activated, and peptides deprotected and added to theactivated COOH groups in a site and sequence-specific manner accordingto the method described below. The solutions used for the couplingreaction were prepared as follows:

Carboxylic Acid Activation Solution:

To prepare the carboxylic acid activation solution, 4% by weight of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 2% by weight ofN-hydroxysuccinimide (NHS) were dissolved in deionized water.

Coupling Photobase Amino Acid Solution 1 (Table 5):

A solution containing the Fmoc-protected amino acid coupling moleculeAlanine was prepared as follows: The polymer poly(methyl methacrylate)(i.e., PMMA) was dissolved in a 1:1 solvent solution ofN-methylpyrrollidone and ethyl lactate. The final concentration of PMMAin solution was 1% by weight. Fmoc-Ala-OH was the coupling molecule andadded to the solution to a final concentration of 2% by weight. Anyother Fmoc protected amino acid can be used in place of Fmoc-Ala-OH forcoupling of another amino acid. Photobase generators1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane and1,3-Bis[(1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane were eachadded to the solution for a final concentration of 1% by weight.

Coupling Photobase Amino Acid Solution 2 (Table 5):

Another solution containing the Fmoc-protected amino acid couplingmolecule Alanine was prepared as follows: The polymer PMMA was dissolvedin the solvent N-methylpyrrollidone. The final concentration of PMMA insolution was 1% by weight. Fmoc-Ala-OH was the coupling molecule andadded to the solution to a final concentration of 2% by weight. Anyother Fmoc protected amino acid can be used in place of Fmoc-Ala-OH forcoupling of another amino acid. Photobase generator1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane was added to thesolution for a final concentration of 1% by weight.

Coupling Photobase Amino Acid Solution 3 (Table 5):

A solution containing the Fmoc-protected amino acid coupling moleculeAlanine was prepared as follows: The polymers PMMA andpolyvinylpyrrolidone were each dissolved in the solventN-methylpyrrollidone. The final concentration of PMMA andpolyvinylpyrrolidone in solution were each 1% by weight. Fmoc-Ala-OH wasthe coupling molecule and added to the solution to a final concentrationof 2% by weight. Any other Fmoc protected amino acid can be used inplace of Fmoc-Ala-OH for coupling of another amino acid. Photobasegenerator 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane wasadded to the solution for a final concentration of 1% by weight.

All Fmoc-protected amino acids were obtained from Anaspec. Polymethylmethacrylate (PMMA) and poly vinyl pyrrollidone were obtained fromPolysciences Inc.

TABLE 5 Photoactive Coupling Formulations Formulation PolymerPhotoactive Compound Coupling Molecule Solvent 1 Polymethyl 1,3-Bis[(2-Fmoc-Ala-OH Ethyl lactate/N- methacrylate nitrobenzyl)oxycarbonyl-methyl-pyrrollidone 4-piperidyl]propane (1:1 by weight) 1,3-Bis[(1-(9-fluorenylmethoxycarbonyl)- 4-piperidyl]propane 2 Polymethyl 1,3-Bis[(2-Fmoc-Ala-OH N-methyl-pyrrollidone methacrylate nitrobenzyl)oxycarbonyl-4-piperidyl]propane 3 Polymethyl 1,3-Bis[(2- Fmoc-Ala-OHN-methyl-pyrrollidone methacrylate nitrobenzyl)oxycarbonyl-4-piperidyl]propane

Solid-Phase N→C Synthesis Methodology

Attachment of a free amino acid to the free carboxylic acid groupattached to the surface of the substrate is shown in FIG. 2. As shown instep 1, the COOH-coated wafer substrate was activated by addingcarboxylic acid activation solution to the surface of the wafer andspinning the wafer to form a layer of carboxylic activation solution onthe surface of the wafer. Carbodiimide in the carboxylic acid activationsolution reacted with the free carboxylic acid groups to generate a freecarbonyl group (e.g., an “activated carboxylic acid group”). Thecarboxylic acid group activation solution was then washed from thesurface of the wafer. As shown in step 2, one of the three couplingphotobase amino acid solutions described above (also see Table 5) wasthen spin-coated onto a wafer at 3000 rpm and baked at 65° Celsius for 1minute on a hot plate. The wafer was then selectively exposed toelectromagnetic radiation at 248 nm and at 80 mJ/cm² using a reticle(Step 3) and then hard baked at 85° Celsius for 90 seconds in a hotplate (Step 4). Fmoc-Ala-OH was deprotected by photoactivated release ofa base from the photobase generator of the coupling photobase amino acidsolution only in the region where it is exposed to radiation. The aminoacid was coupled to the activated carboxylic acid group simultaneouslywith deprotection of the Fmoc-protected amine group. The solution wasthen stripped from the wafer, leaving the newly coupled amino acid boundto the activated carboxylic acid at site-specific locations (Step 5).Steps 2-5 were repeated to couple different amino acids to remainingactivated carboxylic acid groups. After an amino acid had been coupledat each desired location, carboxylic acid group activation as performedin step 1 was optionally repeated to activate carboxylic acid groups onthe entire surface of the array to add another layer of amino acids(cycle of steps 2-5). The process generated sequence-specific peptidechains at specific locations on the substrate. Results obtained forselected sequences are described in further detail below.

20 Mer Homopolymer Synthesis and Coupling Step Efficiency

The photoactive coupling step described above was performed forsynthesizing 20-mer peptides with the following sequences:

Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala

In this example the step yield data for each of the above 20-mer aminoacid sequences was measured. To measure step yield via fluorescence,uncoupled activated carboxylic acids were exposed to a capping solutioncomprising ethanolamine to prevent addition of another amino acid orfluorescein dye molecule. After capping, the fluorescent dye moleculewas coupled to the sequence of amino acids in order to determine thecoupling efficiency according to the following protocol: 5-(Aminomethyl)Fluorescein, Hydrochloride was obtained from Life tech. 0.1 M Boc-Gly-OH(from AAPPTeC), 0.05 M 5-AFH and 0.1 M HoNb (Sigma Aldrich) and 0.1 MEDC (Sigma Aldrich) was dissolved in water along with 5-10% by weightPoly vinyl pyrrollidone (Polysciences). This solution is referred toherein as the “fluorescein coupling solution.” The COOH-coated wafersubstrate comprising capped uncoupled carboxylic acids was activated byadding carboxylic acid activation solution to the surface of the waferand spinning the wafer to form a layer of carboxylic activation solutionon the surface of the wafer. Carbodiimide in the carboxylic acidactivation solution reacted with the free carboxylic acid groups togenerate a free carbonyl group (e.g., an “activated carboxylic acidgroup”). The carboxylic acid group activation solution was then washedaway. The fluorescein coupling solution was then spin coated on thewafer at 2000 rpm to form a coupling dye coat. Next the wafers werebaked at 65° Celsius for 2 minutes and then the fluorescein couplingsolution was washed away with water. This completed the coupling offluorescein dye to measure the proportion of uncapped:capped peptidechains to measure synthesis efficiency. The signal was then read off afluorescence microscope. For all the experiments, the measured signalintensity was directly correlated to the coupling yield. Thedeprotection yield can be calculated by the amount of fluoresceincoupled to the COOH on the substrate after each synthesis step.

The amount of fluorescein dye coupled gives a direct measure of theamount of sequence grown. The formula used to calculate average n-thstep yield (i.e., “F”) was: F=(F_(n)/F₁)/n−1, where F₁ and F_(n) denotesthe fluorescein coupling intensity read out from a fluorescent scannerdevice at the first step and the nth step. The average coupling yield(i.e., average coupling efficiency, or “E”) was calculated using theformula E=10{circumflex over ( )}((log F)/C) where F equals fraction offull length and C=number of couplings=length−1. The step yield at eachstep was calculated by the equation F_(n+1)/F_(n), wherein, after thefirst coupling, n=1, after the second coupling, n=2, and so on. Thecoupling yield at each step was given by the same formula, asfluorescence directly correlates to synthesis efficiency at each step.

FIG. 3A shows a graph of fluorescence signal intensity versus each aminoacid layer. FIG. 3B shows a graph of overall step yield versus eachamino acid layer. Table 6 shows the yield efficiency for each couplingstep. The coupling efficiency of each amino acid was calculated to begreater that 98.5% in each instance across the entire length of the20-mer peptide.

TABLE 6 20-mer homopolymer coupling yield Amino Acid FluorescenceCoupling Efficiency n-th Step Yield 1-mer 61000 1.00000 1.00000 2-mer60363.4 0.98956 0.98956 3-mer 59558.98 0.98812 0.97638 4-mer 588260.98798 0.96436 5-mer 58231 0.98845 0.95461 6-mer 57436.8 0.988030.94159 7-mer 56705.9 0.98791 0.92960 8-mer 56001.23 0.98786 0.918059-mer 55289.1 0.98779 0.90638 10-mer 54576.6 0.98771 0.89470 11-mer53888.2 0.98768 0.88341 12-mer 53212.3 0.98766 0.87233 13-mer 52247.80.98718 0.85652 14-mer 51987.6 0.98778 0.85226 15-mer 51545.7 0.988040.84501 16-mer 50928.9 0.98804 0.83490 17-mer 50526.9 0.98830 0.8283118-mer 49818.6 0.98816 0.81670 19-mer 48959.4 0.98786 0.80261 20-mer48543.4 0.98805 0.79579

12 Mer Heteropolymer Synthesis and Coupling Step Efficiency

The photoactive coupling step described above was performed forsynthesizing up to 12-mer polypeptides. Amino acids used in this examplewere Fmoc-Lys-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Thr-OH, Fmoc-Ser-OH,Fmoc-Asp-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Arg-OH,Fmoc-Val-OH. All amino acids were obtained from Anaspec. These aminoacids were added to the coupling photobase amino acid solution in placeof Fmoc-Ala-OH in the coupling photobase amino acid solutions describedpreviously.

The sequence was synthesized using the carbodiimide activated COOH andFmoc-protected peptide coupling method described above. Fluoresceincoupling was performed to the final product to measure synthesisefficiency as described above.

The 12 mer polypeptide was synthesized according to the following steps:

-   -   1. Lys    -   2. Lys-Leu    -   3. Lys-Leu-Glu    -   4. Lys-Leu-Glu-Arg    -   5. Lys-Leu-Glu-Arg-Ser    -   6. Lys-Leu-Glu-Arg-Ser-Thr    -   7. Lys-Leu-Glu-Arg-Ser-Thr-Val    -   8. Lys-Leu-Glu-Arg-Ser-Thr-Val-Met    -   9. Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile    -   10. Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys    -   11. Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys-Gly    -   12. Lys-Leu-Glu-Arg-Ser-Thr-Val-Met-Ile-Lys-Gly-Asp

The formula used to calculate average n-th step yield (i.e., “F”) was:F=(F_(n)/F₁)/n−1, where F₁ and F_(n) denotes the fluorescein couplingintensity read out from a fluorescent scanner device at the first stepand the nth step. The average coupling yield (i.e., average couplingefficiency, or “E”) was calculated using the formula E=10{circumflexover ( )}((log F)/C) where F equals fraction of full length and C=numberof couplings=length−1. The step yield at each step was calculated by theequation F_(n+1)/F_(n), wherein, after the first coupling, n=1, afterthe second coupling, n=2, and so on. The coupling yield at each step wasgiven by the same formula, as fluorescence directly correlates tosynthesis efficiency at each step.

FIG. 4A shows a graph of fluorescence signal intensity versus each aminoacid layer. FIG. 4B shows a graph of overall step yield for each aminoacid addition. The columns contain the sequence synthesized such thatone amino acid is added in each column.

The coupling efficiency of each amino acid was calculated to be greaterthan 98.5% in each instance across the entire 12-mer peptide and theoverall yield of the full length 12 amino acid polypeptide wascalculated as 86.13%. Table 7 shows the results of the synthesisreaction.

TABLE 7 12-mer heteropolymer yield n-th Amino Peptide Fluore- CouplingStep Acid Sequence scence Efficiency Yield  1-mer K 63987 1.000001.00000  2-mer KL 63276 0.98889 0.98889  3-mer KLE 62431.5 0.987770.97569  4-mer KLER 61504.8 0.98690 0.96121  5-mer KLERS 60648 0.986690.94782  6-mer KLERST 60000 0.98722 0.93769  7-mer KLERSTV 59198 0.987120.92516  8-mer KLERSTVM 58307.5 0.98681 0.91124  9-mer KLERSTVMI 57446.30.98661 0.89778 10-mer KLERSTVMIK 56874.1 0.98699 0.88884 11-merKLERSTVMIKG 56088.8 0.98691 0.87657 12-mer KLERSTVMIKGD 55113.4 0.986520.86132

Example 24: Carboxylic Acid Surface Activation Lifetimes

Wafers with carboxylic acid surfaces were prepared as explained inExample 17 (Trimesic acid coating). Different coupling reagents werethen tested for determining the lifetime of an activated ester.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC] andN-hydroxysuccinimide [NHS] were obtained from Sigma Aldrich.1,3-Diisopropylcarbodiimide [DIC] was obtained from Advanced ChemTech.Hydroxybenzotriazole (HOBt) was obtained from Anaspec.(0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) [HATU] andBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP] were obtained from Aapptec. N,N-Diisopropylethylamine [DIEA] wasobtained from Alfa Aesar.

The wafer was activated with the different combinations of reagentsyielding the following activation solutions: (1) EDC and NHS with 4% byweight of EDC and 2% by weight of NHS were dissolved in deionized waterand reacted with the wafer at room temperature for 10 minutes. The waferwas then washed with deionized water; (2) DIC and HOBt with 4% by weightof DIC and 2% by weight of HOBt were dissolved in NMP and reacted withthe wafer at room temperature for 10 minutes. The wafer was then washedwith NMP; (3) HATU and N,N-Diisopropylethyl-amine (DIEA) with 4% byweight of HATU and 2% by weight of DIEA were dissolved in NMP andreacted with the wafer at room temperature for 8 minutes. The wafer wasthen washed with NMP; and (4) PyBOP and DIEA with 4% by weight of PyBOPand 2% by weight of DIEA were dissolved in NMP and reacted with thewafer at room temperature for 25 minutes. The wafer was then washed withNMP.

After washing the wafers were checked for activation lifetime of eachreagent for different time periods (1 minute, 5 minutes, 20 minutes, 60minutes, 2 hours, 5 hours and 10 hours). For each activation reagent,after each time period, the amount of free carboxylic acid on each arraywas measured by measuring fluorescence intensity using 4′-Aminomethylfluorescein as explained above. Results indicating the lifetime ofactivation of carboxylic acids on the surface of each array are shown inFIG. 5.

PyBOP and DIEA activation ester were susceptible to faster hydrolysis.The other esters showed stability for 5-6 hours. Thus, a singleactivation step can be used for multiple coupling cycles forsequence-specific addition at multiple locations. The above experimentwas also performed with the COOH wafer generated in Example 18 (Melliticacid). Results (not shown) were similar, demonstrating stability ofactivation for 5-6 hours with the above activation solutions (1)-(4).

Example 25: Peptide Synthesis on a COOH Substrate Using Fmoc ProtectedAmino Acids

Wafers with carboxylic acid surfaces were prepared as explained aboveusing trimesic acid (Example 17). Fluorenylmethyloxycarbonyl [Fmoc]protected amino acids were obtained from Anaspec, including Histidine(H), Arginine (R), Serine (S), Valine (V) and Glycine (G). Ethanolaminewas obtained from Sigma Aldrich.

The carboxylic acid surface was activated as follows: 4% by weight ofEDC and 2% by weight of NHS were dissolved in deionized water andreacted with the wafer at room temperature for 10 minutes. The wafer wasthen washed with deionized water.

The deprotection and coupling to the carboxylic acids on the wafer waschecked by binding a sequence-specific antibody to known amino acidsequence (RHSVV). Amino acid coupling was performed as follows: aphotobase coupling solution containing a copolymer (2.5% by weight ofPMMA added to 1.5% by weight of Poly ethylene glycol), 1% by weight ofamino acid and 2.5% by weight of photobase generator was spin-coatedonto a wafer and baked. Next, the wafer was exposed to 248 nm lightusing a reticle and then hard baked. Fmoc was removed from the aminoacid only in the region, where photobase was exposed to light and thedeprotected amino acid was coupled simultaneously. The wafer wasstripped with acetone and isopropyl alcohol [IPA].

After the first layer of amino acid was coupled, ethanolamine was usedfor capping the activated COOH that have not been coupled. This was doneby spin coating a mixture of polymer and ethanolamine in deionized waterand then baking. The wafer was then stripped with deionized water. Theprocess of carboxylic acid activation, deprotection and coupling foreach amino acid at selected sites on the wafer, and capping was repeatedfor coupling each of the next layers of amino acid to the wafer. Twosequences were synthesized on the wafer: RHSVV (natural sequence) andGHSVV (mutant sequence).

After completion of polypeptide synthesis, any protecting groups on theside chains of the polypeptide were removed. Trifluoroacetic Acid [TFA]was obtained from Sigma Aldrich. Pentamethylbenzene [PMB] andThioanisole were obtained from VWR. A hydrogen bromide solution of 33%by weight hydrogen bromide in acetic acid [HBr] was obtained from SigmaAldrich.

The wafer was washed with TFA for 10 minutes. A solution comprising of0.4% by weight PMB and 0.4% by weight thioanisole was dissolved in TFA.After stirring thoroughly, 4% by weight of HBr was added and the waferwas washed with this solution for 60 minutes. The same process wasrepeated again for a further 60 minutes. The wafer was then washed withTFA for 5 minutes, IPA for 5 minutes, then DMF for 5 minutes. The waferwas then neutralized with 5% DIEA in DMF for 5 minutes, then washed withDMF for 5 minutes, and finally washed with IPA for 5 minutes.

Anti-p53 antibody specific to the RHSVV polypeptide and Goat anti-mouseIgG for detection of binding of the anti-p53 antibody to the RHSVVpolypeptide were obtained from ABCAM. TBS Buffer, PBST Buffer and BSAwere obtained from VWR International. The assay to detect polypeptidesynthesis on the peptide array (e.g., the bioassay) was performed asfollows: The chip containing the natural and mutated sequence grown waswashed with methanol for 5 mins followed by washing with TBS Buffer for5 mins. Primary antibody solution containing PBST, 1% BSA, and Anti-p53antibody was incubated on the chip at 370 Celsius for 1 hour. The chipwas then washed with PBST for 5 minutes thrice. (Throughout thisspecification, the absence of a recited temperature indicates that astep was carried out at room temperature, i.e., approximately 23°Celsius.) This was followed by secondary antibody incubation at 370Celsius for 1 hour. The secondary antibody solution contained PBST, 1%BSA, and Goat Anti-mouse IgG. The chip was then washed with PBST for 5minutes thrice. This was followed by washing with deionized water for 5minutes twice. The concentration of anti-p53 antibody was varied and theresults of antibody binding were measured to validate the efficiency ofsynthesis using the single step deprotection and coupling processdescribed above.

Results of antibody binding to the chip comprising RHSVV (normal) versusGHSVV (mutant) sequences are shown in FIG. 6. As shown, antibodyconcentration of 1 pg/ml can be detected using this method and thebinding intensity showed log-linear increase with increasing antibodyconcentration over the lower end of the tested range and plateaus from100 pg/ml to 1 μg/ml.

In the coupling solution, a scavenger can be added to ensure completescavenging of the deprotection product. Examples of such scavengersinclude, but are not limited to, alkyl thiols, such as dithiothreitol,1-propanethiol or 1-decanethiol.

C-terminal amidation can be performed on selective peptides ifnecessary. This process can take place in a solution containing ammoniumchloride, ethylammonium chloride and semicarbazide hydrochloride in thepresence of HATU and DIEA at room temperature.

Example 26: Photobase Generator Compositions

Photobase generator compositions were prepared and tested to determinetheir performance for polypeptide synthesis on an array as describedabove. Each photobase generator composition comprised a photobasegenerator having a structure and general formula as shown in Tables 2and 3. The photobase generators were commercially available orsynthesized as described above.

Preparation of photobase generator compositions was performed asfollows: a mixture of 1-3% by weight of polymethyl methacrylate [PMMA]was added to cyclohexanone and stirred thoroughly for 24 hours. After 24hours stirring, 1.5-5% by weight of photobase generator, depending onthe molecular weight of the photobase generator, was mixed in thesolution and stirred thoroughly for 24 hours. Then, 0.1 M of thesuitable amino acid was added to the solution and stirred for 10 hoursat room temperature.

Wafer with carboxylic acid surfaces were prepared as explained above inExample 17 (Trimesic acid). Different coupling reagents were then testedfor determining the lifetime of an activated ester.1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC] andN-hydroxysuccinimide [NHS] were obtained from Sigma Aldrich.1,3-Diisopropylcarbo-diimide [DIC] was obtained from Advanced ChemTech.Hydroxybenzotriazole (HOBt) was obtained from Anaspec.(0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) [HATU] andBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP] were obtained from Aapptec. N,N-Diisopropylethylamine [DIEA] wasobtained from Alfa Aesar.

Performance of the photobase generator compositions was tested asfollows: Amino acid coupling was performed as described above. Eachcomposition containing a polymer, amino acid and photobase generator wasspin-coated onto a wafer and baked. Next, the wafer was exposed to 248nm radiation and then hard baked. Fmoc amino acids were deprotected onlyin the region where the amino acids were exposed to the radiation. Theamino acid was coupled to the activated carboxylic acid groupsimmediately after exposure to the 248 nm radiation with a list of usedamino acids shown in Table 8. The wafer was then stripped with acetoneand IPA.

TABLE 8 List of amino acids used in photobase generator assay. AminoAcid Description CIT - Citrulline Fmoc-L-Citrulline A - AlanineFmoc-Ala-OH C - cysteine Fmoc-Cys(Bzl)-OH D - aspartic acidFmoc-Asp(Obzl)-OH E - glutamic acid Fmoc-Glu(Obzl)-OH F - phenylalanineFmoc-Phe-OH G - glycine Fmoc-Gly-OH H - histidine Fmoc-His(Trt)-OH I -isoleucine Fmoc-Ile-OH K - lysine Fmoc-Lys(Boc)-OH L - leucineFmoc-Leu-OH M- methionine Fmoc-Met-OH N - asparagine Fmoc-Asn(Trt)-OH P-proline Fmoc-Pro-OH Q - glutamine Fmoc-Gln(Trt)-OH R - arginineFmoc-Arg(Tos)-OH S - serine Fmoc-Ser(Bzl)-OH T - threonineFmoc-Thr(Bzl)-OH V - valine Fmoc-Val-OH W - tryptophan Fmoc-Trp(Boc)-OHY - tryosine Fmoc-Tyr(Bzl)-OH

Ethanolamine was used for capping any activated COOH groups which hadnot been coupled. This was done by spin coating a mixture of polymer,ethanolamine and deionized water onto the wafer and then baking thecoated wafer. The wafer was then stripped with deionized water and thesame process was repeated for coupling the next amino acid.

A sequence of amino acids was synthesized at predetermined locations ona chip by repeating the method above with selected Fmoc-protected aminoacids and polypeptide synthesis performance was determined by usingmeasurements of yield at each step of synthesis. This was done bycoupling one amino acid at a time and finally activating the carboxylicacid groups on the wafer and coupling aminomethyl fluorescein for eachstep yield.

Example 27: Efficiency of Synthesis for Protected and Unprotected AminoAcids

One-step deprotection and coupling was validated in comparison withcoupling of unprotected amino acids and unprotected amino acids in thepresence of a photobase. A wafer with carboxylic acid surface wasprepared as explained above in Example 17 (Trimesic acid).

Amino acids used during synthesis were Citrulline (CIT), Alanine (A),Cysteine (C), Aspartic acid (D), Glutamic acid (E), Phenylalanine (F),Glycine (G), Histidine (H), Isoleucine (I), Lysine (K), Leucine (L),Methionine (M), Asparagine (N), Proline (P), Glutamine (Q), Arginine(R), Serine (S), Threonine (T), Valine (V), Tryptophan (W) and Tryosine(Y). Unprotected amino acids and Fmoc-protected amino acids wereobtained from Anaspec.

The carboxylic acid surface on a wafer was activated with an activationmixture of 4% by weight EDC and 2% by weight NHS dissolved in deionizedwater for 10 minutes. This was followed by washing the wafer withdeionized water for 3 minutes.

Experiment 1 [E1]: Amino Acid coupling was performed as follows: acoupling solution containing a copolymer (2.5% by weight of PMMA addedto 1.5% by weight of Poly Ethylene Glycol) and 1% by weight ofunprotected amino acid was spin-coated onto a wafer and baked. Thereaction resulted in the unprotected amino group in the amino acidcoupling to the activated carboxylic acid present on the surface.Ethanolamine was used for capping any activated COOH groups on thesurface of the wafer, which did not couple to the amino acid. This wasdone by spin coating a mixture of polymer, ethanolamine, and deionizedwater onto the wafer and then baking the coated wafer. The wafer wasthen stripped with deionized water.

Experiment 2 [E2]: Amino Acid coupling was performed as follows: aphotoresist coupling solution containing a copolymer (2.5% by weight ofPMMA added to 1.5% by weight of Poly Ethylene Glycol), 5% by weight ofphotobase generator, and 1% by weight of unprotected amino acid wasspin-coated onto a wafer, baked and the wafer was exposed to 248 nmradiation. The reaction resulted in the unprotected amino group in aminoacid coupling to the activated carboxylic acid present on the surface.Ethanolamine was used for capping any activated COOH groups on thesurface of the wafer which did not couple to the amino acid. This wasdone by spin coating a mixture of polymer, ethanolamine, and deionizedwater onto the wafer and then baking the coated wafer. The wafer wasthen stripped with deionized water. This experiment tested the effect ofbase on the activated ester and tested the effect of base in thecoupling process.

Experiment 3 [E3]: Amino Acid coupling was performed as follows: aphotoresist coupling solution containing a copolymer (2.5% by weight ofPMMA added to 1.5% by weight of Poly Ethylene Glycol), 1% by weight ofunprotected amino acid and 2.5% by weight of photobase generator wasspin-coated onto a wafer and baked. The Fmoc-protected amino acid wasdeprotected, when exposed to 248 nm radiation, allowing the amino groupin amino acid to couple to the activated carboxylic acid present on thesurface with spatial specificity. Ethanolamine was used for capping anyactivated COOH groups on the surface of the wafer which did not coupleto the amino acid. This was done by spin coating a mixture of polymer,ethanolamine, and deionized water onto the wafer and then baking thecoated wafer. The wafer was then stripped with deionized water.

Experiments E1, E2 and E3 were performed for all amino acids. Couplingefficiency for each experiment was determined by adding aminomethylfluorescein directly on the wafer that had been capped before activationas a baseline (CAP+ACT+FLU), and also by activating the wafer andcoupling aminomethyl fluorescein (ACT+FLU) for each experiment. Theresults obtained are shown in FIG. 7.

As seen from the results, the fluorescence intensity appeared relativelyuniform across the all three experiments. Coupling of unprotected aminoacids and Fmoc protected amino acids showed similar coupling efficiency,and coupling under basic conditions present in E2 and E3 did not affectthe yield or the activation ester adversely.

Example 28: Effect of Photobase Generator Concentration on CouplingYield

Concentration of photobase generator in a photoresist solution can be inthe range of 1-30%, preferably in the range 5-15% by weight. The weightpercentage of photobase generator used in the photoresist solution forpeptide coupling was varied to measure the coupling yield. The aminoacid Fmoc-Ala-OH was coupled to the wafer. Amino acid coupling wasperformed as explained above in Example 25 under differentconcentrations of photobase generator in photoresist solution. Unreactedcarboxylic acids were capped using ethanolamine. Carboxylic acid groupsfrom newly coupled alanine were activated as follows: 4% by weight EDCand 2% by weight NHS were dissolved in deionized water for 10 minutesand coated on the wafer. The wafer was then washed with deionized waterfor 3 minutes. Coupling yield was checked by coupling aminomethylfluorescein to newly coupled alanine.

As seen in FIG. 8, low concentration of photobase generator led to lowdeprotection and low coupling yield. Similarly high concentration ofphotobase generator led to good deprotection but poor coupling yield.Optimal concentrations of photobase generator were in the range of5-25%.

Example 29: Coupling of Multiple Amino Acids after a Single ActivationStep

Due to stability of the activated ester of the carboxylic acid for anextended period of time, 25 or more coupling cycles can be completedafter a single activation step to form a complete layer of amino acidsattached to an array. After addition of all amino acids, the wafer wascapped, and the activation, coupling, and capping cycle was optionallyrepeated. The ability to perform multiple couplings at different timesand locations on a wafer after a single activation step was validated bythe following experiment:

The carboxylic acid surface on a wafer was activated by coating with anactivation mixture of 4% by weight EDC and 2% by weight NHS dissolved indeionized water for 10 minutes. This was followed by washing the waferwith deionized water for 3 minutes.

Amino acid coupling was performed as follows: A photoresist couplingsolution containing a polymer, amino acid with amino group protectedwith a light sensitive protecting group was spin-coated onto a wafer andbaked. Next, the wafer was exposed to 248 nm radiation and then hardbaked. Protecting group was removed from the amino acid only in theregion, where the wafer was exposed to the 248 nm radiation. At thisradiation-exposed region, activated carboxylic acid on the surface wascoupled to the deprotected amine group of the amino acid. The wafer wasthen stripped with acetone and IPA. A photoresist coupling solutioncontaining the next amino acid was then used and the same steps asdescribed above were followed to couple this next amino acid to theprevious amino acid.

For consecutive cycles of addition to the activated carboxylic acid, thecapping step described earlier was not performed after each addition,but only after all additions were performed to complete amino acidlayer. Amino acid coupling to the activated carboxylic acid wascontrolled by exposure to light. Coupling did not occur at non-exposedregions of the wafer.

Amino acid coupling yield was calculated for individual activation andcapping and compared to multiple coupling of different amino acid todifferent sites in one activation cycle that was followed by capping.Coupling efficiency was determined by activating and couplingaminomethyl fluorescein to the wafer, where the unbound activatedcarboxylic acids were capped after all coupling steps were completed.

The fluorescent intensity measurements in FIG. 9 showed that individualcoupling of each amino acid in 1 activation cycle was similar tomultiple amino acids coupling in one activation cycle. This demonstratedthat multi-coupling process by our method using a stable activated esterresulted in increased throughput compared to traditional peptidesynthesis methodologies.

Example 30: Peptide Synthesis on a COOH Substrate Using PhotolabileGroup Protected Amino Acids

Wafers with carboxylic acid surfaces were prepared as explained aboveusing trimesic acid (Example 17) and activated with an activationmixture of 4% by weight EDC and 2% by weight NHS dissolved in deionizedwater for 10 minutes. This was followed by washing the carboxyl surfaceof each array with deionized water for 3 minutes.2,2-Dimethyl-3,5-dimethyoxy-benzyloxy-benzocarbonate [DDZ] protectedamino acids were obtained from Anaspec.

Amino Acid coupling was performed as follows: a photoresist couplingsolution containing a copolymer (2.5% by weight of PMMA added to 1.5% byweight of Poly Ethylene Glycol) and 1% by weight of amino acid wasspin-coated onto a wafer and baked. Next, the wafer was selectivelyexposed using a reticle to 248 nm radiation and then hard baked.DDZ-protected amino acids are deprotected only in the region where thewafer was exposed to 248 nm radiation. Deprotected amino acids werecoupled to activated carboxylic acids attached to the wafersimultaneously during bake. Next, the wafer was stripped with acetoneand IPA.

Ethanolamine was used for capping any activated COOH which were notcoupled. This was done by spin coating a mixture of polymer,ethanolamine and deionized water onto the wafer and then baking thecoated wafer. The wafer was then stripped by washing with deionizedwater. The same coupling and capping process was repeated for couplingeach of the next amino acids. All individual amino acids were coupled toselected spots on a chip using a reticle. A range of radiation exposureenergies were used to check coupling yield of each amino acid. This wasdone by coupling one acid at a time and finally activating and couplingaminomethyl fluorescein.

Example 31: Peptide Synthesis Using Fmoc Protected Amino Acids and aPhotoacid Generator

The sequence specificity and final yield of polypeptides on a wafer witha carboxylic acid surface was tested as follows:

Wafers with carboxylic acid surfaces were prepared as explained aboveusing trimesic acid (Example 17). The carboxylic acid surface on a waferwas activated with an activation mixture of 4% by weight EDC and 2% byweight NHS dissolved in deionized water for 10 minutes. This wasfollowed by washing the wafer with deionized water for 3 minutes.

Amino acid coupling was performed as follows: a photoresist couplingsolution containing a copolymer (2.5% by weight of PMMA added to 1.5% byweight of Poly Ethylene Glycol), 1% by weight of Fmoc-protected aminoacid, 5% of N-Boc-piperidine and 2.5% of a photoacid generator wasspin-coated onto a wafer and baked. Next, the wafer was exposed to 248nm radiation and then hard baked. The protecting group Boc was removedfrom piperidine only in the region where it is exposed. Piperidineremoved Fmoc protection from the amino acid and the activated carboxylicacid on the surface was coupled to the amine group of the deprotectedamino acid in the exposed regions. The wafer was then stripped withacetone and IPA. For multiple couplings, the cycle of activation andcoupling described above is repeated with a new photoresist couplingsolution containing the next amino acid.

The accuracy and efficiency of peptide synthesis using this method wasmeasured by synthesizing the sequence RHSVV (Natural Sequence) and itsmutated sequence GHSVV (Mutant Sequence) on a carboxylic acid waferusing the method described above.

After synthesis, the side chains of the amino acid were deprotectedaccording to the following protocol: Trifluoroacetic Acid [TFA] wasobtained from Sigma Aldrich. Pentamethylbenzene [PMB] and thioanisolewas obtained from VWR. A solution of 33% by weight hydrogen bromidedissolved in acetic acid [HBr] was obtained from Sigma Aldrich.

The wafer was washed with TFA for 10 minutes. A solution comprising 0.4%by weight of PMB and 0.4% thioanisole was dissolved in TFA. Afterstirring thoroughly, 4% of HBr was added to the solution and the waferwas washed twice with this solution for 60 minutes each. The wafer wasthen washed with TFA for 5 minutes, IPA for 5 minutes, then DMF for 5minutes. The wafer was then neutralized with 5% DIEA in DMF for 5minutes, then washed with DMF for 5 minutes, and finally washed with IPAfor 5 minutes.

The sequence specific binding of antibodies to the chip was performed asfollows: The chips containing the synthesized natural and mutatedsequences were washed with methanol for 5 minutes, then were washed withTBS Buffer for 5 minutes. The primary antibody solution containing PBST,1% BSA and anti-p53 antibody was incubated on the surface of the waferat 37 Celsius for 1 hour. The chip was washed with PBST for 5 minutesthrice. This was followed by incubating the chip with secondary antibodysolution at 370 Celsius for 1 hour. The secondary antibody containedPBST, 1% BSA, and Goat anti-mouse IgG. The chip was washed with PBST for5 minutes thrice. This was followed by washing with deionized watertwice for 5 minutes each. The concentration of anti-p53 antibody wasvaried to validate the efficiency of coupling using the above process.

The results as shown in FIG. 10 were consistent with the sequences grownusing one-step deprotection and coupling validation process above. Thebinding of antibody concentration of 1 pg/mL to the correct sequence canbe detected using this method. The binding intensity showed a log-linearincrease with increasing antibody concentration over the lower end ofthe range tested and plateaus from 100 pg/mL to 1 μg/mL. Thisdemonstrated the use photoacid generator instead of a photobasegenerator in a coupling solution using a protected piperidine base.

Example 32: Photoinduced Carbodiimides for Peptide and ProteinMicroarray Preparation

This example did not rely on amino protecting groups and enabledcarboxylic acids attached to the surface of an array to be selectivelyactivated using photoinduced carbodiimide chemistry with selective photoirradiation through a photomask or automatic exposure method like amicromirror. The general activation chemistry for a tetrazole thione toform a carbodiimide is given in Scheme 2. After activation of thecarboxylic acid groups by the photoactivated carbodiimide, amino acidsor peptide chains having an unprotected amine group were added to thearray and coupled to the activated carboxylic acid.

Process Flow for Preparing a Protein Array:

Wafers were prepared with COOH substrate as described in Example 13. Oneof three activation solutions was prepared as described below.4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,and1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thionewere obtained from Sigma Aldrich Inc. Polyvinyl pyrrollidone wasobtained from Polysciences Inc.

Photoactivated Carboxylic Acid Activation Solution 1: 2.5% by weight of4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione wasdissolved in 95% DI water along with 2.5% by weight of polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

Photoactivated Carboxylic Acid Activation Solution 2: 2.5% by weight of1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thionewas dissolved in 95% DI water along with 2.5% by weight of Polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

Photoactivated Carboxylic Acid Activation Solution 3: 2.5% by weight of1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thionewas dissolved in 95% DI water along with 2.5% by weight of Polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

One of the above activation solutions comprising a thione was coatedonto the wafer and baked at 85° Celsius for 90 seconds. The coat wasexposed at 248 nm at 10-100 mJ/cm² using a photomask to choose regionsto couple protein. In the exposed regions, the thione was converted intoa carbodiimide (see, e.g., Scheme 2). Photoactivated conversion of1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thioneto 1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-carbodiimide ofactivation solution 3 occurred at 248 nm and at 10-100 mJ/cm² (see,e.g., Scheme 3). The carbodiimide activated the carboxylic acid groupsattached to the array by forming carbonyl groups ready to bind to anamino group. The activation solution was then washed from the chip, andthe carboxylic acid groups remain activated for a certain amount oftime. Protein coupling solution comprising 50 μg/mL of TNF-alphadissolved in 5% polyvinyl pyrrollidone in deionized water was preparedand was coated on the wafer at 2000 rpm. Then, the wafers were baked at37° Celsius for 5 minutes to complete the TNF alpha coupling to theactivated carboxylic acid comprising regions of the chip. The processabove was repeated using IL-6 in place of TNF alpha and activatingdifferent regions on the chip. The complete process for site-specificactivation of carboxylic acid groups via site-specific photoactivationof carbodiimide, and the attachment of protein to the activated sites,is depicted in FIG. 11.

To confirm attachment of TNF alpha and IL-6 to the correct locations onthe chip, anti-TNF alpha and Anti IL-6 antibodies were added to thechip. All antibodies and buffer solutions were obtained from LifeTechnologies. The assay was performed as follows: anti-TNF alpha andAnti IL-6 antibodies were diluted 1:1000 in PBST buffer. Chips werewashed in PBST buffer thrice for 5 minutes. The antibody solution wasadded to the chip and incubated for 1 hour at 37° Celsius in the dark.The chips were then washed with PBST buffer thrice for 5 min followed bydeionized water thrice for 5 minutes. The chips were then scanned in afluorescent scanner.

Data for the two proteins on the array is shown in FIG. 12. SignalIntensities are represented in a scale from 0 to 65000. Binding to eachprotein was performed in quadruplicate (i.e., features 1-4). As shown inFIG. 12, TNF-alpha and IL-6 proteins each bound to their respectivesites that were photoactivated by the method above before addition ofthe proteins. Therefore, photoactivated carbodiimide chemistry forattachment of polypeptides to activated carboxylic acid groups providedlocation-specific attachment of IL-6 and TNF-alpha to the array.

Example 33: Photoinduced Carbodiimides for Peptide Synthesis

In this example, method of C→N synthesis of peptides on a chip arrayusing site-specific photoactivated carbodiimide activation of freecarboxylic acid groups and attachment of unprotected amino acids tophotoactivated carboxylic acid sites was performed. Wafers with COOHgroups were prepared as explained in Example 13. The solutions used forthe coupling reaction were as follows:

One of three activation solutions was prepared as described below.4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,and1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thionewere obtained from Sigma Aldrich Inc. Polyvinyl pyrrollidone wasobtained from Polysciences Inc.

Activation Solution 1: 2.5% by weight of4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione wasdissolved in 95% DI water along with 2.5% by weight of polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

Activation Solution 2: 2.5% by weight of1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thionewas dissolved in 95% DI water along with 2.5% by weight of polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

Activation Solution 3: 2.5% by weight of1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thionewas dissolved in 95% DI water along with 2.5% by weight of polyvinylpyrrollidone and spun in a magnetic stirrer overnight to dissolvecompletely.

Coupling Solutions were Prepared as Follows:

Coupling amino acid solution 1: A solution containing the amino acidcoupling molecule alanine was prepared as follows: The polymerpoly(methyl methacrylate) (i.e., PMMA) was dissolved in a 1:1 solventsolution of N-methylpyrrollidone and ethyl lactate. The finalconcentration of PMMA in solution was 1% by weight. Alanine was thecoupling molecule and added to the solution for a final concentration of2% by weight. Any other amino acid may be used in place of alanine forcoupling of this other amino acid.

Coupling amino acid solution 2: Another solution containing the aminoacid coupling molecule alanine was prepared as follows: The polymer PMMAwas dissolved in the solvent N-methylpyrrollidone. The finalconcentration of PMMA in solution was 1% by weight. Alanine was thecoupling molecule and added to the solution for a final concentration of2% by weight. Any other amino acid may be used in place of alanine forcoupling of this other amino acid.

Coupling amino acid solution 3: A solution containing the amino acidcoupling molecule alanine was prepared as follows: The polymers PMMA andpolyvinylpyrrolidone were each dissolved in the solventN-methylpyrrollidone. The final concentration of PMMA andpolyvinylpyrrolidone in solution were each 1% by weight. Alanine was thecoupling molecule and added to the solution for a final concentration of2% by weight. Any other amino acid may be used in place of alanine forcoupling of this other amino acid.

Polymethyl methacrylate (PMMA) and poly vinyl pyrrollidone were obtainedfrom Polysciences Inc.

Solid-Phase N→C Synthesis Methodology

One of the above activation solutions comprising a thione was coatedonto the wafer and baked at 85° Celsius for 90 seconds. The coat wasexposed at 248 nm at 10-100 mJ/cm² using a photomask to choose regionsto couple the protein to. In the exposed regions, the thione wasconverted into a carbodiimide (see, e.g., Scheme 2). Photoactivatedconversion of1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thioneto 1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-carbodiimide ofactivation solution 3 occurred at 248 nm and at 10-100 mJ/cm²(see, e.g.,Scheme 3). The carbodiimide activated the carboxylic acid groupsattached to the array by forming carbonyl groups ready to bind to anamino group. The activation solution was then washed from the chip, andthe carboxylic acid groups remained activated for at least 15 minutes.

One of the three amino acid coupling solutions described above was thenlayered on top of the wafer to allow reaction between the activatedcarboxylic acid groups and the amino acids. The amino acid was coupledto the activated carboxylic acid group. The solution was then washed,leaving the newly coupled amino acid bound to the activated carboxylicacid at site-specific location. The process was repeated to add desiredamino acids at reticle-specified activated carboxylic acid locations togenerate sequence-specific peptide chains at specific locations on thesubstrate.

1.-211. (canceled)
 212. A method of attaching a coupling molecule to asubstrate, comprising: obtaining a substrate comprising a plurality ofcarboxylic acid groups for linking to a coupling molecule; contactingsaid substrate with a carboxylic acid activating compound formulationcomprising a carbodiimide precursor, wherein said carbodiimide precursorconverts to a carbodiimide upon exposure to electromagnetic radiation ata defined wavelength; selectively exposing said carboxylic acidactivating compound formulation to the electromagnetic radiation,thereby generating said carbodiimide and activating the carboxylic acidgroups to form carbonyl groups on said substrate at a selectivelyexposed area; contacting said substrate with a coupling formulationcomprising a coupling molecule and a solvent; coupling the couplingmolecule to at least one of said plurality of carbonyl groups at saidselectively exposed area.
 213. The method of claim 212, wherein saidcarbodiimide precursor is formula (I):

wherein R is selected from a group comprising hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, andsubstituted or unsubstituted heterocyclyl, and R further comprises awater-solubilizing group; and R′ is substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstituted aryl,substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocyclyl.
 214. The method of claim 213, wherein saidcarbodiimide precursor is a thione or a tetrazole thione.
 215. Themethod of claim 214, wherein said tetrazole thione is selected from thegroup consisting of:1-(3-(diethylamino)-propyl)-4-(2-methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione,4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione,4-cyclohexyl-1H-tetrazole-5(4H)-thione, and1-phenyl-4-(piperidinomethyl)-tetrazole-5(4H)-thione.
 216. The method ofclaim 212, wherein said defined wavelength is 248 nm or 193 nm.
 217. Themethod of claim 212, wherein said carboxylic acid activating compoundformulation or said coupling formulation further comprises a polymer.218. The method of claim 217, wherein said polymer is poly(methylmethacrylate), polyvinyl pyrrolidone, or polyvinyl alcohol.
 219. Themethod of claim 212, wherein said carboxylic acid activating compoundformulation and said coupling formulation are present on the substratewhen said substrate is exposed to said electromagnetic radiation. 220.The method of claim 212, wherein said coupling step is performedmultiple times at different selectively exposed areas on said substrate.221. The method of claim 212, wherein said coupling step has a couplingefficiency of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,98.5%, or 99%.
 222. The method of claim 212, wherein said couplingmolecule is an amino acid, a protein, or a polypeptide.
 223. The methodof claim 212, wherein said coupling molecule comprises a protected aminegroup,
 224. The method of claim 224, wherein said amine group isprotected by Fmoc.
 225. The method of claim 212, further comprisingrepeating said method to produce a desired polypeptide at said at leastone carbonyl group.
 226. The method of claim 212, wherein said substratecomprises a porous layer, and optionally wherein said substratecomprising a plurality of attachment sites extending in multipledimensions from the surface of said porous layer within and around saidporous layer.
 227. The method of claim 212, wherein said substratecomprises a planar layer comprising a metal and having an upper surfaceand a lower surface; and a plurality of pillars operatively coupled tothe layer in positionally-defined locations, wherein each pillar has aplanar surface extended from the layer, wherein the distance between thesurface of each pillar and the upper surface of the layer is between1,000-5,000 angstroms, wherein the surface of each pillar is parallel tothe upper surface of the layer, and wherein the plurality of pillars arepresent at a density of greater than 10,000/cm2, and wherein theattachment site is coupled to the upper surface of the pillar.
 228. Acarboxylic acid activating formulation, comprising: a carboxylic acidactivating compound and a solvent, wherein said carboxylic acidactivating compound is a carbodiimide precursor of formula (I):

wherein R is selected from a group comprising hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, andsubstituted or unsubstituted heterocyclyl, and R further comprises awater-solubilizing group; and R′ is substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstituted aryl,substituted or unsubstituted cycloalkyl, and substituted orunsubstituted heterocyclyl.
 229. The carboxylic acid activatingformulation of claim 228, wherein said carbodiimide precursor is athione or a tetrazole thione.
 230. The carboxylic acid activatingformulation of claim 229, wherein said thione is selected from the groupconsisting of:1-(3-(diethylamino)-propyl)-4-(2-methoxyphenyl)-1,4-dihydro-5H-tetrazole-5-thione;4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-(3-(dimethylamino)propyl)-4-ethyl-1,4-dihydro-5H-tetrazole-5-thione,1,4-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-1,4-dihydro-5H-tetrazole-5-thione,4-cyclohexyl-1H-tetrazole-5(4H)-thione, and1-phenyl-4-(piperidinomethyl)-tetrazole-5(4H)-thione; or
 231. Thecarboxylic acid activating formulation of claim 228, wherein saidcarbodiimide precursor converts to carbodiimide upon exposure toelectromagnetic radiation at a defined wavelength.
 232. The carboxylicacid activating formulation of claim 231, wherein said definedwavelength is 248 nm or 193 nm.